HK40011663B

HK40011663B - Somatic cell production system

Info

Publication number: HK40011663B
Application number: HK62020000973.1A
Authority: HK
Inventors: 田边刚士; 须藤健太; 平出亮二
Original assignee: 田边刚士; 爱平世股份有限公司
Filing date: 2017-02-27
Publication date: 2021-06-04

Description

Somatic cell production system

Technical Field

The present invention relates to a somatic cell induction technology, and more particularly, to a somatic cell production system.

Background

Embryonic Stem cells (ES cells) are Stem cells established from early embryos of humans or mice. ES cells have pluripotency that can differentiate into all the cells present in an organism. Human ES cells are now used in cell transplantation therapy for a number of diseases such as parkinson's disease, juvenile onset diabetes, and leukemia. However, there are also obstacles in the transplantation of ES cells. In particular, transplantation of ES cells may cause the same immunological rejection as that caused after unsuccessful organ transplantation. Furthermore, there are many cases where the ES cells established by disrupting human embryos are used in ethical opinion or against.

Under this background, professor kyoto university teaches the expression of the gene sequence by combining 4 genes: oct3/4, Klf4, c-Myc, and Sox2 were introduced into somatic cells, and induced pluripotent stem cells (iPS cells) were successfully established. Therefore, professor in mountains acquired the nobel physiological or medical award of 2012 (see patent document 1, for example). iPS cells are ideal pluripotent cells without rejection or ethical issues. Therefore, the iPS cells are expected to be applied to cell transplantation therapy. In recent years, a technique has been established in which a specific gene is introduced into a cell to produce another cell from the specific cell. Such a technique is expected to be applied to transplantation medicine or drug screening as with iPS cells.

Currently, there are many methods to transform iPS cells into somatic cells. However, in order to use iPS cells for transplantation therapy, it is important to establish a method for inducing differentiation with good efficiency of iPS cells. Specifically, it is necessary to establish a technique for inducing differentiation of iPS cells into somatic cells, so that the efficiency and accuracy of differentiation induction are improved and the prepared somatic cells can withstand transplantation therapy, for example, for functionality.

The method of inducing differentiation from iPS cells or embryonic stem cells (ES cells) into somatic cells was performed in the following manner: the production process is simulated by combining hormones or growth factors, which determine the properties of the cells, and low molecular compounds, and changing their quantitative ratio or concentration over time. However, it is difficult to fully mimic the production process in a cuvette and is inefficient. In addition, human beings require a very long differentiation induction period, for example, 3 months or more for producing mature nerves, as compared with the induction of somatic differentiation in mice. In addition, there are problems that the efficiency of differentiation induction greatly varies depending on the ES/iPS cell line, and the properties of the induced somatic cells are not uniform.

Specifically, it was confirmed that cells induced by differentiation of human ES/iPS cells using a method using hormones or chemicals were somatic cells at an early stage in a fetal stage. The differentiation induction of human mature somatic cells is extremely difficult and requires long-term culture over several months. However, in drug development or transplantation medicine for developing an individual who has completed development, it is very important to produce somatic cells that match the maturity of the individual.

Furthermore, there are various types of cells in nerve cells, but these types of nerves cannot be uniformly differentiated and induced from ES/iPS cells by a method using hormones or chemical substances. Therefore, specific drug development and screening specific to neural subtypes cannot be performed. Therefore, the efficiency of drug development screening is low. In transplantation medicine, it is also impossible to concentrate only certain cells of a transplantation disease.

In this regard, the following methods are proposed: genes that specify the properties of specific somatic cells were directly introduced into ES/iPS cells using viruses to create target somatic cells. In contrast to the method using hormones or chemicals, the method using viruses can specifically produce mature nerve cells in a very short time, for example, 2 weeks. Furthermore, if a nerve cell is created by introducing a specific gene, only excitatory nerves can be uniformly obtained, for example. Therefore, it is possible to perform development and screening of specific nerve subtype-specific drugs, and it is also possible to concentrate only certain specific cells of a transplant disease in transplant medicine.

Furthermore, in the case of differentiating iPS cells into somatic cells, there is a concern that: undifferentiated iPS cells remained in the differentiated somatic cells. Therefore, a method of differentiating somatic cells into other somatic cells not via iPS cells has also been established. Specifically, methods for differentiating fibroblasts into cardiomyocytes or neurocytes have been developed. These methods are called direct reprogramming, and do not involve pluripotent stem cells such as iPS cells, and therefore there is no risk of undifferentiated pluripotent cells remaining at the time of transplantation.

Documents of the prior art

Patent document

Patent document 1: japanese patent No. 4183742

Disclosure of Invention

Problems to be solved by the invention

Somatic cells can be established by introducing an inducer such as a gene into the cells, and then cultured by amplification and, if necessary, frozen for storage. However, for example, there are problems as described below in the production of clinical somatic cells (e.g., GLP, GMP grade) and in industrialization.

1) Cost of

Clinical somatic cells need to be prepared and stored in a clean room. However, the cost of maintaining the required level of cleanliness is very high. Therefore, it is necessary to produce clinical somatic cells at a low cost, which is a serious obstacle to industrialization.

2) Quality of product

The series of operations from establishment to preservation of somatic cells is complicated, and many manual operations are required. Moreover, the production of somatic cells sometimes depends on individual skills. Therefore, variations in the quality of clinical somatic cells may occur depending on the manufacturer or experimental lot.

3) Time of day

In order to prevent cross-contamination of cells with other than a specific donor in a clean room, it takes a predetermined time to prepare clinical somatic cells derived from only one person in the clean room. Moreover, establishment and quality evaluation of clinical somatic cells require a long time. However, since only one individual clinical somatic cell is prepared in a clean room at a time, it takes a very long time to prepare clinical somatic cells of many customers.

4) Man-made material

As described above, in the present situation, the production of clinical somatic cells requires many manual operations. However, there are few technical personnel who have the techniques necessary to be able to produce clinical somatic cells.

In view of the above, an object of the present invention is to provide a somatic cell production system capable of producing somatic cells. The somatic cells are not limited to clinical somatic cells.

Means for solving the problems

According to an aspect of the present invention, there is provided a somatic cell production system including: a pre-cell introduction liquid-feeding path through which a solution containing a pre-cell to be introduced passes; a factor introducing device connected to the pre-introduction cell liquid-feeding passage, for introducing a somatic cell-inducing factor into the pre-introduction cells to produce induced factor-introduced cells; and a cell preparation device for culturing the inducer-introduced cells to prepare somatic cells.

The somatic cell production system may further include a housing that houses the pre-introduction cell feeding channel, the factor introduction device, and the cell production device.

In the above somatic cell production system, the somatic cells prepared by introducing the somatic cell-inducing factor may not include pluripotent stem cells. The somatic cells prepared by introducing the somatic cell-inducing factor may include differentiated cells. The somatic cells prepared by introducing the somatic cell-inducing factor may include adult stem cells. Adult stem cells refer to adult stem cells or tissue stem cells. The somatic cells prepared by introducing the somatic cell-inducing factor may include neural cells. The somatic cells prepared by introducing the somatic cell-inducing factor may include fibroblasts. The somatic cell prepared by introducing the somatic cell-inducing factor may include a cardiomyocyte, a keratinocyte (keratinocyte), or a retinal cell.

In the above somatic cell production system, the cells before introduction may include pluripotent stem cells. Pluripotent stem cells may include ES cells and iPS cells. The cells prior to introduction may comprise adult stem cells. The cells prior to introduction may comprise differentiated somatic cells. The cells prior to introduction may comprise blood cells. The cells prior to introduction may comprise fibroblasts.

In the above somatic cell production system, the cell production device may include: a somatic cell culture device for culturing the induced factor-introduced cells produced in the factor-introducing device; and an amplification culture device for performing amplification culture on the somatic cells established in the somatic cell culture device, wherein the somatic cell culture device is provided with a 1 st medium supply device for supplying a medium to the cells for introducing the induction factors, and the amplification culture device is provided with a 2 nd medium supply device for supplying a medium to the somatic cells.

In the above somatic cell production system, the somatic cell culture apparatus may further include a drug supply device that supplies a solution containing a drug that kills cells to which the drug-resistant factor has not been introduced.

In the above somatic cell production system, the factor introducing device may include: a factor-introducing part connected to the pre-introduction cell liquid-feeding channel; a factor storage unit for storing a somatic cell-inducing factor; a factor-delivering channel for allowing the somatic cell-inducing factor to flow from the factor-storing part to the pre-introduction cell-delivering channel or the factor-introducing part; a pump for causing the liquid in the factor liquid feeding passage to flow.

In the above somatic cell production system, the somatic cell-inducing factor may be DNA, RNA, or protein.

In the factor introducing part of the somatic cell production system, a somatic cell-inducing factor can be introduced into the cells before introduction by RNA lipofection.

In the above somatic cell production system, the somatic cell-inducing factor may be implanted into the carrier. The vector may be a Sendai virus vector.

The somatic cell production system may further include a packaging device for packaging the somatic cells produced by the cell production device, and the packaging device may be housed in a case.

The somatic cell production system may further include a solution displacer including a cylindrical member and a liquid-permeable filter disposed inside the cylindrical member, wherein the cylindrical member is provided with: a somatic cell introduction hole for introducing a solution containing somatic cells prepared in the cell preparation device onto the liquid-permeable filter; a substitution solution introduction hole for introducing the substitution solution onto the liquid-permeable filter; a somatic cell discharge hole for discharging a substitution solution containing somatic cells onto the liquid-permeable filter; and a waste liquid outflow hole for allowing the solution permeating the filter to flow out.

The above somatic cell production system may further comprise a waste liquid feeding passage connected to the waste liquid outflow hole of the solution displacer, and the solution in the waste liquid feeding passage is allowed to flow when the solution containing the somatic cells is discarded, and the solution in the waste liquid feeding passage is not allowed to flow when the somatic cells are dispersed in the substitution solution.

In the above somatic cell production system, the substitution solution may be a frozen storage solution.

The somatic cell production system may further include a separation device for separating the pre-introduced cells from the blood, and the solution containing the pre-introduced cells separated by the separation device may pass through the pre-introduced cell liquid-feeding passage.

ADVANTAGEOUS EFFECTS OF INVENTION

According to the present invention, a somatic cell production system capable of producing somatic cells can be provided.

Drawings

FIG. 1 is a schematic view of a somatic cell production system according to an embodiment of the present invention.

FIG. 2 is a schematic view of a somatic cell production system according to an embodiment of the present invention.

FIG. 3 is a schematic cross-sectional view of an example of an introduced cell liquid-feeding channel in the somatic cell production system according to the embodiment of the present invention.

FIG. 4 is a schematic cross-sectional view of an example of an introduced cell liquid-feeding channel in the somatic cell production system according to the embodiment of the present invention.

FIG. 5 is a schematic view of a culture bag used in the somatic cell production system according to the embodiment of the present invention.

Fig. 6 is a schematic diagram of a solution displacer according to an embodiment of the invention.

FIG. 7 is a schematic view of a somatic cell production system according to an embodiment of the present invention.

FIG. 8 is a photograph of the cells of example 1.

FIG. 9 is a photograph of the cells of example 1.

FIG. 10 is a graph showing the ratio of transfection efficiency to survival rate in example 1.

FIG. 11 is a photograph of the cells of example 2.

FIG. 12 is a graph showing the ratio of TUJ-1 positive cells in example 2.

FIG. 13 is a graph showing the ratio of TUJ-1 positive cells in example 2.

FIG. 14 is a schematic representation of the transfection method of example 3.

FIG. 15 is a photograph of the cells of example 3.

Detailed Description

Hereinafter, embodiments of the present invention will be described. In the description of the drawings, the same or similar reference characters denote the same or similar parts. The drawings are schematic drawings. Therefore, specific dimensions and the like should be determined in accordance with the following description. It is to be noted that the drawings naturally include portions having different dimensional relationships or ratios from each other.

Additionally, the disclosure is incorporated into U.S. provisional application (62/356,199), and has issued an invention licensed for foreign application.

As shown in fig. 1, the somatic cell production system according to the embodiment of the present invention includes: a pre-cell introduction liquid-feeding channel 20 through which a solution containing pre-cells to be introduced passes; a factor introducing device 30 connected to the pre-introduction cell liquid-feeding channel 20 for introducing a somatic cell-inducing factor into the pre-introduction cells to produce induced factor-introduced cells; a cell preparation device 40 for culturing the induction factor-introduced cells to prepare somatic cells; and a housing 200 for accommodating the pre-introduction cell liquid feeding path 20, the factor introducing device 30, and the cell preparing apparatus 40.

The cells before introduction are, for example, pluripotent stem cells. The pluripotent stem cells may be ES cells or iPS cells. Alternatively, the cells before introduction are, for example, differentiated cells. Examples of the differentiated cells include somatic cells differentiated from adult stem cells, blood cells, and fibroblasts. Adult stem cells are also known as adult stem cells or tissue stem cells.

Somatic cells prepared by introducing a somatic cell-inducing factor do not include pluripotent stem cells. Somatic cells prepared by introducing a somatic cell-inducing factor are differentiated cells. Examples of the differentiated cells include: adult stem cells, neural line cells, fibroblasts, cardiac muscle cells, liver cells, retina cells, corneal cells, blood cells, keratinocytes (keratinocytes), chondrocytes, and the like. The neural line cell may be any of a neural cell, a neural stem cell and a neuronal precursor cell. The nerve cell may be any of an inhibitory nerve cell, an excitatory nerve cell and a dopamine-producing nerve cell. Alternatively, the neural line cells may be motor nerve cells, oligodendrocyte precursor cells, oligodendrocytes, and the like. The neural line cells may be MAP2 positive or β -IIITubulin positive.

The somatic cell manufacturing system may further comprisePreparing: an air cleaning device for cleaning the air in the housing 200; a temperature management device that manages the temperature of the gas in the casing 200; and a carbon dioxide concentration control device for controlling carbon dioxide (CO) in the gas in the housing 200₂) And (4) concentration. The air cleaning device may also contain a cleanliness sensor for monitoring the cleanliness of the gas within the housing 200. The air cleaning device cleans air in the casing 200 using, for example, a hepa (high Efficiency Particulate air) filter, an ulpa (ultra Low networking air) filter, or the like. The air cleaning device, for example, sets the cleanliness of the air in the housing 200 to a level of ISO1 to ISO6 in ISO standard 14644-1. The temperature management device may also contain a temperature sensor for monitoring the temperature of the gas within the housing 200. CO 2₂The concentration management device also comprises CO₂Concentration sensor for monitoring CO of gas in housing 200₂And (4) concentration.

The case 200 is provided with a door, for example, and the inside is completely closed in a state where the door is closed, so that the cleanness, temperature, and CO of the inside air can be maintained₂The concentration was kept constant. The case 200 is preferably transparent so that the state of the internal device can be observed from the outside. The housing 200 may be a glove box (glove box) in which gloves such as rubber gloves are integrally formed.

The housing 200 may be provided with an inlet port communicating with the pre-introduction cell liquid feeding path 20. A door or the like may be provided in the opening. Alternatively, the inlet may be sealed with a detachable sealing material. The cells before introduction enter the cell-before-introduction liquid-feeding channel 20 from the introduction port. Alternatively, a pre-introduction cell storage container for storing the pre-introduction cells may be disposed in the housing 200, and may communicate with the pre-introduction cell liquid-feeding passage 20.

Further alternatively, the somatic cell production system may further include a separation device 10 disposed in the casing 200 and configured to separate the cells before introduction from the blood, as shown in fig. 2. In this case, the pre-introduction cell liquid-feeding channel 20 is connected to the separation apparatus 10. The solution containing the pre-cell to be introduced separated in the separation apparatus 10 passes through the pre-cell to be introduced liquid sending passage 20.

The separation device 10 within the housing 200 receives a vial of, for example, human blood. The separation apparatus 10 includes an anti-coagulant tank for storing an anti-coagulant such as ethylenediaminetetraacetic acid (EDTA), heparin, and a biological preparation-based blood storage solution a liquid (ACD-a liquid, talocene). The separation apparatus 10 adds the anti-coagulant from the anti-coagulant tank to the human blood using a pump or the like.

The separation apparatus 10 is provided with a separation reagent tank for storing a reagent for separating monocytes, such as Ficoll-Paque PREMIUM (registered trademark, Nippon general electric medical Co., Ltd.). The separation apparatus 10 dispenses the reagent for separating mononuclear cells from the reagent tank for separating mononuclear cells by 5mL each time into, for example, 2 tubes of 15mL using a pump or the like. In addition, a resin bag may be used instead of the tube.

The separation device 10 is provided with a buffer tank for storing a buffer such as Phosphate Buffered Saline (PBS). The separation apparatus 10 dilutes 5mL of buffer solution by adding the buffer solution from a buffer solution tank to 5mL of human blood, for example, using a pump or the like. Furthermore, the separation apparatus 10 adds the diluted human blood to the reagent for separating monocytes in the tube by 5mL each time using a pump or the like.

The separation device 10 further includes a centrifuge capable of setting a temperature. The centrifuge is set to, for example, 18 ℃. The separation apparatus 10 uses a mobile apparatus or the like, and places a tube containing a reagent for separating monocytes, human blood, and the like on a holder of a centrifuge. The centrifuge performs centrifugation of the solution in the tube for 30 minutes at, for example, 400 Xg. The resin bag may be centrifuged instead of the tube.

After centrifugation, the separation device 10 recovers the white turbid intermediate layer of monocytes in the solution in the tube with a pump or the like. The separation apparatus 10 sends the collected suspension of monocytes to the pre-introduction cell liquid-sending passage 20 by using a pump or the like. Alternatively, further, the separation apparatus 10 adds, for example, 12mL of PBS to 2mL of the recovered monocyte solution, and places the tube on the holder of the centrifuge. The centrifuge performs centrifugation of the solution in the tube for 10 minutes at, for example, 200 Xg.

After centrifugation, the separator 10 removes the supernatant of the solution in the tube by suction using a pump or the like, and 3mL of a monocyte culture medium such as X-VIVO 10 (registered trademark, Lonza Japan) is added to the monocyte solution in the tube and suspended. The blood cells may be cultured without a feeder layer using a substrate film such as matrigel (corning), CELLstart (registered trademark, thermo fisher), or lamin 511 (nippi). The separation apparatus 10 sends the suspension of monocytes as pre-introduction cells to the pre-introduction cell liquid sending channel 20 by using a pump or the like. Alternatively, the separation device 10 may separate monocytes from blood using a dialysis membrane. In addition, when using a pre-introduced cell prepared in advance, the separation apparatus 10 may not be used.

The separation device 10 may separate cells suitable for induction by a method other than centrifugation. For example, if the cell to be isolated is a T cell, panning can be used to isolate a cell positive for any of CD3, CD4, and CD 8. If the cells to be isolated are vascular endothelial precursor cells, panning may be used to isolate cells that are positive for CD 34. If the cells to be isolated are B cells, panning may be used to isolate cells positive for any of CD10, CD19, CD 20. Furthermore, the separation is not limited to the panning, and the separation may be performed by a magnetic cell separation Method (MACS), a flow cytometer, or the like.

Poly-HEMA (poly 2-hydroxymethacrylate) to which the cells before introduction do not adhere can be introduced on the inner wall coating of the cell liquid-feeding channel 20 before introduction, and is rendered non-adherent to the cells. Alternatively, the material of the pre-introduction cell liquid-feeding channel 20 may be a material to which the pre-introduction cells are less likely to adhere. The material introduced into the anterior cell liquid-feeding channel 20 is, for example, CO having good thermal conductivity₂A permeable material, whereby the conditions in the pre-cell liquid-feeding channel 20 are adjusted to the temperature and CO regulated in the housing 200₂The concentrations were equal. Further, from the viewpoint of preventing contamination, a backflow prevention valve may be provided in the pre-introduction cell liquid feeding path 20.

The inducer solution feeding mechanism 21 in the housing 200 includes an inducer introducing reagent tank for storing an inducer introducing reagent solution or the like, for example. The inducer solution feeding mechanism 21 feeds the inducer introducing reagent solution to the pre-introduction cell solution feeding path 20 or the factor introducing device 30 in the housing 200 by using a micro-pump or the like so that the pre-introduction cells are suspended in the inducer introducing reagent solution.

The inducer reagent solution such as the gene transfer reagent solution includes, for example, a set containing somatic inducer RNA, an RNA transfection solution, and a medium for RNA transfection. RNA transfection includes RNA lipofection. The kit of somatic cell induction factor RNA comprises, for example, 100ng each of mRNA of ASCL1, mRNA of Myt1L, and mRNA of neurogenin2(Ngn 2). Ngn2(neurogenin2) is a switch protein required for the differentiation of neural lineage cells.

The somatic cell induction factor RNA may further contain mRNA corresponding to the drug resistance gene. Examples of the drug include puromycin, neomycin, blasticidin, G418, hygromycin, bleomycin and other antibiotics. Cells into which mRNA corresponding to the drug resistance gene has been introduced exhibit drug resistance. The somatic cell-inducing factor RNA comprises, for example, Ngn2-T2A-Puro mRNA (Trilink). Cells transfected with Ngn2-T2A-Puro mRNA (Trilink) produced neurogenin2(Ngn2) and showed puromycin resistance.

mRNA can also be capped with Anti-Reverse Cap Analog (ARCA), polyadenylated, and replaced with 5-methylcytidine and pseudouridine. 5-methylcytidine and pseudouridine reduced the ability of the antibody to recognize mRNA.

The RNA transfection solution comprises, for example, small interfering RNA (sirna) or lipofection agents. Lipofection reagents for RNA include lipofection reagents for siRNA and lipofection reagents for mRNA. More specifically, lipofectin for RNA may be Lipofectamine (registered trademark) RNAiMAX (registered trademark) siemens technology, Lipofectamine (registered trademark) 2000, Lipofectamine (registered trademark) 3000, neofecton System (registered trademark) messenger max (sermmer trademark) RNA transfection reagent (Stemfect), NextFect (registered trademark) RNA transfection reagent (biorientic), Amaxa (registered trademark) Human T cellNucleofector (registered trademark) kit (Lonza corporation, VAPA-1002), Amaxa (registered trademark) Human CD34cellNucleofector kit (registered trademark) mRNA (registered trademark), mRNA technology (registered trademark) mRNA, and the like.

For example, the factor introducing device 30 may suspend the cells in a culture solution after introducing the somatic cell-inducing factor into the cells. Factor introduction device 30 may perform multiple transfections of somatic cell inducing factors. For example, after a predetermined time such as 24 hours after the introduction of the somatic cell-inducing factor into the cells, the medium may be replaced, and the cells may be transfected again with the somatic cell-inducing factor. The step of transfecting the somatic cell-inducing factor into the cell and culturing the cell for a predetermined period of time may be repeated a plurality of times, for example, 2 to 4 times.

In lipofection of somatic cell-inducing factor RNA, the number of cells per 1 well is 1X 10, for example, in the case of using 12 microtiter plates⁴1 to 10⁸5 x 10 pieces of⁴1 to 10⁶1, or 1 × 10⁵To 5 x 10⁵And (4) respectively. In addition, the bottom area of the 1-hole is 4cm². The amount of the somatic cell-inducing factor RNA upon lipofection of the somatic cell-inducing factor RNA is 200ng to 5000ng, 400ng to 2000ng, or 500ng to 1000ng per 1 time. The amount of the lipofection reagent at the time of lipofection of the somatic cell induction factor RNA is 0.1. mu.L to 100. mu.L, 1. mu.L to 50. mu.L, or 1.5. mu.L to 10. mu.L.

The medium used for lipofection of somatic cell induction factor RNA is, for example, a low serum medium such as Opti-MEM (registered trademark, Gibco). The medium used at and before lipofection of somatic cell induction factor RNA may contain B18R protein. The B18R protein moderates the innate anti-viral response of cells. The B18R protein is useful for inhibiting cell death associated with an immune response when RNA is inserted into a cell. However, in the case of differentiating cells into somatic cells in a short time, the medium may not contain the B18R protein, or may contain the B18R protein at a lower concentration of 0.01% to 1%.

The animal cells differentiate into somatic cells within 10 days, within 9 days, within 8 days, or within 7 days after lipofection of the somatic cell-inducing factor RNA. When the somatic cell thus prepared is a neural cell, whether or not the somatic cell has differentiated into the neural cell can be confirmed by whether or not β -IIITubulin, MAP2, or PsA-NCAM is positive. beta-IIITubulin, MAP2, PsA-NCAM, and vGlu are markers for identifying nerve cells, and are structural proteins of microtubules in the nerve cell process.

Alternatively, the inducer-introducing agent solution such as the gene-introducing agent solution may include, for example, a Sendai virus vector solution. The RNA derived from Sendai virus is not integrated into the host DNA, but the target gene can be introduced into the host. The Sendai virus vector set contains mRNA of ASCL1, mRNA of Myt1L, and mRNA of Ngn2, for example, so that the MOI (multiplicity of infection) is 0.01 to 1000, 0.1 to 1, or 1 to 10. The inducer sendai virus vector may contain mRNA corresponding to the drug resistance gene. The inducer RNA contained in the Sendai virus vector contains, for example, Ngn2-T2A-Puro mRNA (Trilink). In addition, Sendai virus can be introduced into cells 1 time.

The factor introducing device 30 sends a solution containing cells into which an induction factor has been introduced (induction factor-introduced cells) to the introduced cell liquid sending channel 31 using a pump or the like.

The inner wall of the introduced cell liquid-feeding passage 31 in the housing 200 may be coated with poly-HEMA to which the inducer is not attached, so that the inducer is non-attached to the cells. Alternatively, a material that is difficult to adhere to the inducer-introduced cells may be used as the material to be introduced into the cell liquid-feeding channel 31. In addition, for example, the material introduced into the cell liquid-feeding channel 31 has good thermal conductivity and CO₂A permeable material, whereby the conditions for introduction into the cell liquid-feeding channel 31 are adjusted to the temperature and CO regulated in the case 200₂The concentrations were equal. Further, from the viewpoint of preventing contamination, a backflow prevention valve may be provided in the introduced cell liquid feeding path 31. Further, as shown in FIG. 3, one or more folds may be provided inside the introduced cell liquid feeding path 31 so that the inner diameter thereof is intermittently changed. Further alternatively, as shown in FIG. 4, the inner diameter of the introduced cell liquid-feeding channel 31 may be changed intermittently.

As shown in FIGS. 1 and 2, the cell preparation apparatus 40 connected to the introduced cell liquid-feeding channel 31 includes: a somatic cell culture apparatus 50 for culturing the induced factor-introduced cells produced in the factor-introducing apparatus 30; a 1 st dividing means 60 for dividing a cell mass (cell colony) consisting of somatic cells established in the somatic cell culture apparatus 50 into a plurality of cell masses; an amplification culture device 70 for performing amplification culture on somatic cells; a 2 nd dividing means 80 for dividing a cell mass comprising somatic cells expanded and cultured in the expansion culture apparatus 70 into a plurality of cell masses; the somatic cell transfer mechanism 90 sequentially transfers the somatic cells to the packaging apparatus 100. However, for example, when a cell mass is not formed or when it is not necessary to divide the cell mass, the 1 st division mechanism 60 and the 2 nd division mechanism 80 may be omitted.

The somatic cell culture apparatus 50 may include a culture container such as a microtiter plate, a bag, or a tube. The somatic cell culture apparatus 50 may further include a liquid transfer device. The somatic cell culture apparatus 50 receives a solution containing a somatic cell-inducing factor-introduced cell from the introduction cell liquid-feeding passage 31, and dispenses the solution into the culture vessel by using a pipetting machine.

In the case of differentiating cells into neural cells, the somatic cell culture apparatus 50, after introducing an inducing factor into the cells into the culture vessel, adds N3 medium (DMEM/F12, 25. mu.g/mL insulin, 50. mu.g/mL human transferrin, 30nmol/L sodium selenite, 20nmol/L progesterone, 100nmol/L putrescine) as a neural differentiation medium to the culture vessel on, for example, days 1 to 7. ROCK inhibitor (seleck) may also be added to the medium at a concentration of 10. mu. mol/L for several days.

After the somatic cell culture apparatus 50 dispenses the inducer-introduced cells into the culture vessel, the medium is changed, for example, on day 9, and then the medium is changed until the target cells such as the neural cells are observed. Further, the term "medium replacement" also includes replacement of a part of the medium or replenishment of the medium.

In the somatic cell culture apparatus 50, a drug that causes cells into which no drug-resistant factor has been introduced to die may also be selected. When the somatic cell-inducing factor RNA contains mRNA corresponding to a drug-resistant gene, the inducer that exhibits drug resistance can be selectively introduced into cells and continued to survive by supplying a solution containing a drug to the culture vessel. For example, when the somatic cell-inducing factor RNA contains mRNA corresponding to a puromycin resistance gene, cells transfected with lipids can be exposed to puromycin to cause cells other than the cells into which the somatic cell-inducing factor RNA has been introduced to die, thereby screening the cells into which the somatic cell-inducing factor RNA has been introduced. The agent may also be contained in the culture medium. The concentration of the drug is, for example, 2 mg/L.

In the somatic cell culture apparatus 50, the inducer-introduced cells may be cultured for a predetermined period of time using a medium containing a drug that causes the death of cells into which the drug-resistant factor has not been introduced, and then cultured using a medium containing no drug.

When the target somatic cells are formed, the somatic cell culture apparatus 50 recovers the somatic cells by using a pipetting machine. Further, the somatic cell culture apparatus 50 places the container containing the recovered somatic cells in an incubator at 37 ℃ with CO₂Somatic cells were reacted with trypsin instead of recombinase at 5% for 10 min. In addition, when the cell mass is physically disrupted, trypsin may not be used instead of the recombinase. For example, the somatic cell culture apparatus 50 breaks up a cell mass of somatic cells by pipetting with a pipetting machine. Further alternatively, the somatic cell culture apparatus 50 may break the cell mass by passing the cell mass through a conduit provided with a filter or a conduit whose inner diameter is intermittently changed in the same manner as the introduction of the cell liquid feeding path 31 shown in FIG. 3 or FIG. 4.

In the case of, for example, nerve induction, thereafter, the somatic cell culture apparatus 50 adds the neural differentiation medium as described above to a solution containing a cell mass of disrupted somatic cells.

In addition, the culture in the somatic cell culture apparatus 50 may be performed not in a microtiter plate but in a bag. The bag may be CO₂Permeability. The culture may be an adhesion culture or a suspension culture. In the case of suspension culture, agitation culture may also be performed. Further, the culture in the somatic cell culture apparatus 50 may also be a hanging drop culture.

The somatic cell culture apparatus 50 may further include the 1 st medium supply device for supplying a medium containing a culture medium to a culture vessel such as a microtiter plate, a bag, or a tube. The 1 st medium supplying apparatus may collect the culture medium in the culture vessel, filter the culture medium using a filter or a dialysis membrane, and reuse the purified culture medium. In addition, growth factors and the like may be added to the reused culture solution. The somatic cell culture apparatus 50 may further include a drug supply device for supplying a solution containing a drug for killing cells into which a drug-resistant factor has not been introduced into the culture container. Further, the somatic cell culture apparatus 50 may further include a temperature control device for controlling the temperature of the culture medium, a pH control device for controlling the pH of the culture medium, a humidity control device for controlling the humidity in the vicinity of the culture medium, and the like.

In the somatic cell culture apparatus 50, for example, cells may be placed in a culture solution permeable bag 301 such as a dialysis membrane shown in FIG. 5, the culture solution permeable bag 301 may be placed in a culture solution non-permeable bag 302, and a culture solution may be placed in the bags 301 and 302. The bag 302 may be CO₂Permeability, which may or may not be CO₂Permeability. The somatic cell culture apparatus 50 may be configured such that a plurality of bags 302 containing fresh culture medium are prepared in advance, and the bag 302 placed outside the bag 301 containing cells is replaced with the bag 302 containing fresh culture medium at predetermined intervals.

The somatic cell culture apparatus 50 shown in FIGS. 1 and 2 is connected to the 1 st somatic cell feeding channel 51. The somatic cell culture apparatus 50 sends a solution containing somatic cells to the 1 st somatic cell feeding channel 51 using a pump or the like. The 1 st somatic cell liquid-feeding channel 51 is connected to a branch channel having an inner diameter that allows only cells that are induced to have a size smaller than a predetermined size to pass through, and is configured to remove cells that are not induced to have a size equal to or larger than the predetermined size.

It is also possible to make the inner wall of the 1 st somatic cell feeding channel 51 non-adherent to poly-HEMA without causing adhesion of somatic cells. Alternatively, the material of the 1 st somatic cell feeding channel 51 may beMaterials to which somatic cells are difficult to attach are used. The material of the 1 st somatic cell feeding channel 51 used was CO having good thermal conductivity₂A permeable material, whereby the conditions in the 1 st somatic cell liquid-feeding passage 51 are adjusted to the temperature and CO regulated in the housing 200₂The concentrations were equal. Further, from the viewpoint of preventing contamination, a backflow prevention valve may be provided in the 1 st somatic cell feeding passage 51.

The 1 st somatic cell feeding channel 51 is connected to the 1 st division mechanism 60. The 1 st division mechanism 60 includes, for example, a mesh. When the cell mass contained in the solution passes through the mesh due to the water pressure, the cell mass is divided into a plurality of cell masses each having a size of each hole of the mesh. For example, if the size of each hole of the mesh is uniform, the size of the plurality of divided cell masses becomes substantially uniform. Alternatively, the 1 st division mechanism 60 may further include a nozzle. For example, by finely processing the inside of a nozzle having a substantially conical shape into a stepped shape, a cell mass contained in a solution is divided into a plurality of cell masses when passing through the nozzle.

The 1 st division mechanism 60 is connected to an amplification culture apparatus 70. The solution containing the cell mass of the somatic cells divided in the 1 st division mechanism 60 is sent to the amplification culture apparatus 70. In addition, when the cell mass is not formed, the 1 st division mechanism 60 may be omitted. In this case, the 1 st somatic cell feeding channel 51 is connected to the amplification culture apparatus 70.

The amplification culture apparatus 70 may house a microtiter plate inside, for example. The amplification culture apparatus 70 is provided with a liquid transfer machine. The amplification culture apparatus 70 receives the solution containing the somatic cells from the 1 st division mechanism 60 or the 1 st somatic cell feeding channel 51, and dispenses the solution into the wells by using a pipetting machine. The amplification culture apparatus 70 dispenses somatic cells into wells, and then CO is performed at 37 ℃₂In the case of 5%, the somatic cells are cultured for about 8 days, for example. The amplification culture apparatus 70 is suitably changed in medium.

When the cell mass is formed, the amplification culture apparatus 70 adds trypsin, such as TrypLE Select (registered trademark, life technologies), instead of the recombinase, to the cell mass. Further, the amplification culture apparatus 70 raises the temperature of the container containing the cell massAt 37 ℃ and CO₂At 5% the cell pellet was reacted with trypsin instead of recombinase for 1 min. In addition, when the cell mass is physically disrupted, trypsin may not be used instead of the recombinase. For example, the amplification culture apparatus 70 disrupts the cell mass by pipetting with a pipetting machine. Further alternatively, the amplification culture apparatus 70 may break the cell mass by passing the cell mass through a conduit provided with a filter or a conduit intermittently changing the inner diameter in the same manner as the introduction cell liquid-feeding passage 31 shown in FIG. 3 or FIG. 4. Thereafter, the amplification culture apparatus 70 shown in FIG. 1 and FIG. 2 adds a culture medium such as a culture medium for maintenance culture to the solution containing the cell mass. Further, in the case of the adhesion culture, the amplification culture apparatus 70 peels the cell mass from the container by an automatic cell scraper or the like, and sends the solution containing the cell mass to the 1 st division mechanism 60 via the amplification culture solution sending passage 71.

In addition, the culture in the amplification culture apparatus 70 may be performed not in a microtiter plate but in a bag or a tube. The bag or tube may be CO₂Permeability. The culture may be an attachment culture, a suspension culture, or a hanging drop culture. In the case of suspension culture, agitation culture may also be performed.

The amplification culture apparatus 70 may further include a 2 nd medium supply apparatus for supplying a culture medium to a culture vessel such as a microtiter plate, a bag, or a tube. The 2 nd medium replenishing apparatus may further collect the culture solution in the culture vessel, filter the culture solution using a filter or a dialysis membrane, and reuse the purified culture solution. In addition, growth factors and the like may be added to the reused culture solution. The amplification culture apparatus 70 may further include a temperature control device for controlling the temperature of the culture medium, a humidity control device for controlling the humidity in the vicinity of the culture medium, and the like.

In the amplification culture apparatus 70, for example, cells may be placed in a culture solution permeable bag 301 such as a dialysis membrane as shown in FIG. 5, the culture solution permeable bag 301 may be placed in a culture solution non-permeable bag 302, and a culture solution may be placed in the bags 301 and 302. The bag 302 may also be CO₂Through the use ofAnd (4) sex. The amplification culture apparatus 70 may be configured such that a plurality of bags 302 containing fresh culture medium are prepared in advance, and the bag 302 placed outside the bag 301 containing cells is replaced with a bag 302 containing fresh culture medium at predetermined intervals.

The somatic cell production system shown in fig. 1 and 2 may further include an amplification culture imaging device for imaging the culture in the somatic cell culture device 50 and the amplification culture device 70. Here, if a colorless medium is used as the medium used in the somatic cell culture apparatus 50 and the amplification culture apparatus 70, it becomes possible to suppress the diffuse reflection or autofluorescence that may be generated in the case of using a colored medium. However, in order to confirm the pH of the medium, a pH indicator such as phenol red may be included. In addition, since the shape, size, and the like of the cells are different between the induced cells and the cells that are not induced, the somatic cell production system may further include an induction state monitoring device, and the proportion of the induced cells may be calculated by imaging the cells in the somatic cell culture device 50 and the amplification culture device 70. Alternatively, the induction state monitoring device may determine the ratio of cells to be induced by an antibody immunostaining method or an RNA extraction method. Further, the somatic cell production system may further include an uninduced cell removal device for removing uninduced cells by a magnetic cell separation method, a flow cytometer, or the like.

The cell masses divided by the 1 st division mechanism 60 shown in FIGS. 1 and 2 are again cultured in the amplification culture apparatus 70. The division of the cell mass by the 1 st division means 60 and the culture of the somatic cells in the amplification culture apparatus 70 are repeated until a desired cell amount is obtained. As described above, when the cell mass is not formed, the 1 st division mechanism 60 may be omitted.

The 2 nd somatic cell feeding path 72 is connected to the amplification culture apparatus 70. The amplification culture apparatus 70 sends the solution containing the somatic cells to be subjected to amplification culture to the 2 nd somatic cell feeding passage 72 by using a pump or the like. In which, in the case of suspension culture, no detachment is required. The 2 nd somatic cell liquid-feeding passage 72 may be connected to a branch flow passage having an inner diameter through which only cells that are induced to have a size smaller than a predetermined size pass, and through which cells that are not induced to have a size larger than the predetermined size are removed.

It is also possible to make the inner wall of the 2 nd somatic cell feeding passage 72 non-adherent with poly-HEMA that does not adhere to the somatic cells. Alternatively, the material of the 2 nd somatic cell liquid-feeding passage 72 may be a material to which somatic cells are less likely to adhere. The material for the 2 nd somatic cell feeding passage 72 has good thermal conductivity and CO₂A permeable material, whereby the conditions in the 2 nd somatic cell liquid-feeding passage 72 are adjusted to the temperature and CO managed in the casing 200₂The concentrations were equal. Further, from the viewpoint of preventing contamination, a backflow prevention valve may be provided in the 2 nd somatic cell feeding passage 72.

The 2 nd somatic cell feeding channel 72 is connected to the 2 nd dividing mechanism 80. The 2 nd division mechanism 80 includes, for example, a mesh. When the cell mass contained in the solution passes through the mesh due to the water pressure, the cell mass is divided into a plurality of cell masses each having a size of each hole of the mesh. For example, if the size of each hole of the mesh is uniform, the size of the plurality of divided cell masses becomes substantially uniform. Alternatively, the 2 nd division mechanism 80 may be provided with a nozzle. For example, by finely processing the inside of a nozzle having a substantially conical shape into a stepped shape, a cell mass contained in a solution is divided into a plurality of cell masses when passing through the nozzle.

The somatic cell transfer mechanism 90 is connected to the 2 nd division mechanism 80 shown in fig. 2, and the somatic cells are sequentially transferred to the sealing device 100. In addition, when the cell mass is not formed, the 2 nd division mechanism 80 may be omitted. In this case, the 2 nd somatic cell feeding passage 72 is connected to the somatic cell transfer mechanism 90.

The pre-encapsulation cell channel 91 is connected between the somatic cell transfer mechanism 90 and the encapsulation device 100 in the housing 200. The somatic cell transfer mechanism 90 sequentially transfers somatic cells to the sealing device 100 through the pre-sealing cell channel 91 using a pump or the like.

Poly-HEMA to which the somatic cells do not adhere may be coated on the cell channel 91 before encapsulation. Alternatively, the material of the cell channel 91 before encapsulation may be a material to which somatic cells are less likely to adhere. The material for the pre-encapsulation cell flow path 91 is excellent in thermal conductivity and CO₂A permeable material, whereby the conditions in the cell channel 91 before sealing are adjusted to the temperature and CO managed in the case 200₂The concentrations were equal. From the viewpoint of preventing contamination, a backflow prevention valve may be provided in the pre-encapsulation cell flow path 91.

The pre-encapsulation cell channel 91 is connected to a frozen preservative solution feeding mechanism 110. The frozen preservation solution feeding mechanism 110 feeds the frozen preservation solution to the pre-encapsulation cell channel 91. Therefore, in the pre-encapsulation cell channel 91, the somatic cells are suspended in the cell freezing and storing solution.

The packaging device 100 sequentially freezes the somatic cells delivered through the pre-packaging cell channel 91. For example, when receiving somatic cells, the packaging device 100 puts the somatic cells into a cryopreservation vessel such as a cryopreservation tube, and instantaneously freezes a solution containing the somatic cells to, for example, -80 ℃. Since it takes time to freeze if a cryopreservation vessel having a small surface area per unit volume is used, it is preferable to use a cryopreservation vessel having a large surface area per unit volume. By using a cryopreservation vessel having a large surface area per unit volume, the survival rate of cells after thawing can be improved. Examples of the shape of the cryopreservation vessel include a capillary tube shape and a spherical shape, but the shape is not limited to these shapes. Further, depending on the desired survival rate of the cells after thawing, instantaneous freezing may not be necessary.

The freezing is performed by, for example, a Vitrification (Vitrification) method. In this case, DAP213(COSMO BIO Co., Ltd.) and Freezing Medium (Reversal Co., Ltd.) can be used as the cell Freezing and preserving solution. The freezing can be performed by a usual method other than the vitrification method. In this case, cryo-preservative solution may be used such as CryoDefex-Stem Cell (R & D Systems Co.), STEM-CELLBANKER (registered trademark, Nippon Kagaku Co., Ltd.). The freezing may be performed by liquid nitrogen or by a peltier element. If the Peltier element is used, temperature variation can be controlled, and temperature unevenness can be suppressed. The packaging device 100 carries out the cryopreservation vessel out of the casing 200. In the case where frozen cells are used clinically, the cryopreservation vessel is preferably a fully closed system. However, the packaging device 100 may be packaged in a storage container without freezing the somatic cells.

Alternatively, the solvent of the solution containing the somatic cells may be replaced from the culture medium to the frozen storage solution in the packaging apparatus 100 by using the solution replacement device 101 shown in fig. 6. A filter 102 is provided inside the solution displacer 101, and the filter 102 has a pore on the bottom surface through which somatic cells do not permeate. The solution displacer 101 is provided with: a somatic cell introduction hole having a 1 st liquid feeding channel 103 connected to the filter 102 inside for feeding a culture medium containing somatic cells; a substitution solution introduction hole having a 2 nd liquid feeding channel 104 connected to the filter 102 inside for feeding the frozen liquid containing no somatic cells; the somatic cell discharge hole is connected to a 1 st discharge channel 105 for discharging a frozen liquid containing somatic cells from the filter 102 inside. The solution displacer 101 is further provided with a waste liquid outflow hole, and a 2 nd discharge channel 106 for discharging the solution having passed through the filter 102 is connected thereto. The 1 st liquid feeding channel 103, the 2 nd liquid feeding channel 104, the 1 st discharge channel 105, and the 2 nd discharge channel 106 may be formed of a tube or the like.

First, as shown in fig. 6 (a) and 6 (b), the medium containing the somatic cells is placed into the solution displacer 101 from the 1 st liquid feeding channel 103 in a state where the flow of the solution in the 2 nd discharge channel 106 is stopped. Next, as shown in FIG. 6 (c), the culture medium is discharged from the solution displacer 101 in a state where the solution in the 2 nd discharge channel 106 is allowed to flow. At this time, as shown in fig. 6 (d), somatic cells remain on the filter 102. Further, as shown in FIG. 6 (e) and FIG. 6 (f), in a state where the flow of the solution in the 2 nd discharge channel 106 is stopped, the frozen preservation solution is put into the solution displacer 101 from the 2 nd liquid feeding channel 104, and the somatic cells are dispersed in the frozen preservation solution. Thereafter, as shown in fig. 6 (g), the frozen preservation solution containing the somatic cells is discharged from the first discharge channel 1 105. The cryopreservation solution containing the somatic cells is sent to a cryopreservation vessel or the like through the 1 st discharge channel 105.

The somatic cell production system shown in fig. 1 and 2 may further include a sterilization device for sterilizing the inside of the casing 200. The sterilization device may be a dry heat sterilization device. In this case, the wirings of the electric devices used, such as the separation device 10, the pre-introduction cell liquid feeding path 20, the induction factor liquid feeding mechanism 21, the factor introduction device 30, the cell preparation device 40, and the packaging device 100, are preferably heat-resistant wirings. Alternatively, the sterilization apparatus may discharge a sterilization gas such as ozone gas, hydrogen peroxide gas, or formalin gas into the casing 200 to sterilize the inside of the casing 200.

The somatic cell production system can also transmit the operation records of the separation device 10, the pre-introduction cell liquid-feeding passage 20, the induction factor liquid-feeding mechanism 21, the factor introduction device 30, the cell production device 40, the packaging device 100, and the like, and the images photographed by the photographing device to an external server by wire or wireless. Further, the external server may control the separation apparatus 10, the induction factor liquid feeding mechanism 21, the factor introducing apparatus 30, the cell preparation apparatus 40, the packaging apparatus 100, and the like of the somatic cell production system based on a Standard Operation Program (SOP), monitor whether or not each apparatus is operated based on the SOP, and automatically generate an operation record of each apparatus.

According to the somatic cell production system described above, somatic cells can be automatically induced.

The somatic cell production system according to the embodiment is not limited to the structure shown in fig. 1 and 2. For example, in the system for producing somatic cells according to the embodiment shown in fig. 7, blood is sent from the blood storing part 201 to the monocyte separation part 203 via the blood sending channel 202. For example, a tube can be used for the blood storing part 201 and the monocyte isolating part 203. The blood feeding path 202 is, for example, a resin tube or a silicon tube. The same applies to other liquid feeding passages described later. The blood storing part 201 may be attached with an identifier such as a barcode to manage blood information. The pump 204 is used for liquid feeding. The pump 204 may use a positive displacement pump. Examples of positive displacement pumps include: a reciprocating pump including a piston pump, a plunger pump, and a diaphragm pump, or a rotary pump including a gear pump, a vane pump, and a screw pump. Examples of the diaphragm pump include a tube pump and a piezoelectric (piezo) pump. Examples of the tube pump include a peristaltic pump (registered trademark, ATTO Co., Ltd.), and RP-Q1 and RP-TX (high sand electric industry Co., Ltd.). Examples of the piezoelectric pump include SDMP304, SDP306, SDM320, and APP-20KG (high sand electric industries, Ltd.). Furthermore, a microfluidic chip module (high sand electric industries co., ltd) incorporating various pumps may also be used. If a closed type pump such as a peristaltic pump (registered trademark), a tube pump, or a diaphragm pump is used, the liquid can be delivered without directly contacting the blood in the blood liquid delivery path 202. The same applies to other pumps described later. Alternatively, the pump 204, and the pumps 207, 216, 222, 225, 234, 242, and 252 described later may be syringe pumps. Even a pump other than the sealed pump can be reused by heat sterilization or the like.

The erythrocyte coagulation medium is fed from the storage 205 of the separating medium to the monocyte separation part 203 via the liquid feeding path 206 and the pump 207. For example, a tube can be used as the separating agent storage 205. The separating agent storage 205 may be attached with an identifier such as a barcode to manage the information of the separating agent. Examples of the hemagglutinating agent include HetaSep (registered trademark, stem cell Technologies), hemagglutinating agent (nipulo (NIPRO)) and the like. In the monocyte separation section 203, the erythrocytes are sedimented by the erythrocyte aggregating agent, and the monocytes are separated. The supernatant containing the monocytes in the monocyte separation part 203 is sent to the monocyte purification filter 210 via the monocyte liquid feeding passage 208 and the pump 209. In the monocyte purification filter 210, components other than monocytes are removed to obtain a solution containing monocytes as pre-introduction cells. As the monocyte purification filter 210, there can be used Purecell (registered trademark, PALL), Cellsorba E (Asahi Kasei corporation), Sepacell PL (Asahi Kasei corporation), Adacolumn (registered trademark, JIMRO), a separation bag (Nipulo Kasei corporation), and the like.

In fig. 7, the monocyte separation unit 203, the separating agent storage unit 205, the monocyte purification filter 210, and the pumps 204, 207, 209 constitute a separation apparatus. However, in the case where cells before introduction prepared in advance are used as described above, the separation apparatus may be omitted.

The solution containing the cells before introduction is sent to the factor-introducing part 213 via the cells before introduction liquid-sending passage 211 and the pump 212. The factor introducing part 213 may be a tube, for example. The somatic cell-inducing factor is sent from the factor storage part 214 containing the somatic cell-inducing factor to the factor introduction part 213 via the factor liquid-sending passage 215 and the pump 216. The factor storage unit 214 may be a tube, for example. The information on the somatic cell-inducing factor may be managed by attaching an identifier such as a barcode to the factor storage unit 214. The factor storage unit 214, the pump 216, and the like constitute an inducer solution feeding mechanism. In the factor introducing part 213 as a factor introducing device, a somatic cell inducing factor is introduced into cells by, for example, RNA lipofection, and induced factor-introduced cells are produced. However, the transfection method of the inducing factor is not limited to the RNA lipofection method. For example, Sendai virus vectors containing somatic cell-inducing factors can also be used. Alternatively, the somatic cell-inducing factor may be a protein. Moreover, transfection of the inducing factors can be performed several times and for up to several days.

The inducer-introduced cells are sent to a somatic cell culture vessel 219, which is a part of the cell production apparatus, via an introduced cell liquid-sending passage 217 and a pump 218. The introduced cell liquid-feeding channel 217 is, for example, temperature-permeable and CO₂Permeability. The first days after the introduction of the somatic cell-inducing factor into the cells, the cell culture medium containing the drug is supplied to the somatic cell culture container 219 from the cell culture medium storage unit 220 containing the cell culture medium containing the drug via the medium feeding path 221 and the pump 222. The agent-containing cell culture medium contains an agent that kills cells into which the drug-resistant factor has not been introduced. The medium liquid feeding path 221 is, for example, temperature-permeable and CO₂Permeability. The drug-containing cell culture medium storage unit 220 may be attached with an identifier such as a barcode to manage information on the drug-containing cell culture medium. The chemical-containing cell culture medium storage unit 220, the medium feeding path 221, and the pump 222 constitute a medium supply device.

Thereafter, the somatic cell culture medium is supplied to the somatic cell culture container 219 from the somatic cell culture medium storage unit 223 containing the somatic cell culture medium suitable for the target somatic cell via the medium supply passage 224 and the pump 225. Can also be used in somatic cell cultureThe medium storage unit 223 manages information on the somatic cell culture medium by attaching an identifier such as a barcode. The medium feeding path 224 is, for example, temperature-permeable and CO₂Permeability. The somatic cell culture medium storage section 223, the medium feeding path 224, and the pump 225 constitute a medium replenishment device.

The cell culture medium storage unit 220 and the somatic cell culture medium storage unit 223 can be refrigerated at a low temperature such as 4 ℃ in the refrigerated storage unit 259, for example. The medium supplied from the drug-containing cell culture medium storage unit 220 and the somatic cell culture medium storage unit 223 may be heated to 37 ℃ by a heater outside the cryopreservation unit 259, for example, and then supplied to the incubator. Alternatively, the temperature of the culture medium stored at a low temperature may be set so that the temperature of the medium in the vicinity of the liquid feeding path is increased to 37 ℃ when the medium enters the liquid feeding path. The medium that has become old in the somatic cell culture device 219 is sent to the waste liquid storage 228 via the waste liquid sending passage 226 and the pump 227. The waste liquid storage 228 may be attached with an identifier such as a barcode to manage information on waste liquid.

The somatic cells cultured in the somatic cell culture device 219 are sent to the 1 st amplification culture device 232, which is a part of the cell production apparatus, through the introduced cell feeding passage 229, the pump 230, and the optional cell clump cutter 231. The cell mass is passed through a cell mass divider 231, thereby dividing the cell mass into smaller cell masses. In the case where the cell mass is not formed, the cell mass divider 231 may be omitted. The somatic cell culture medium is supplied from the somatic cell culture medium storage section 223 containing the somatic cell culture medium to the 1 st amplification culture vessel 232 via the medium feeding passage 233 and the pump 234. The introduced cell liquid-feeding channel 229 and the medium liquid-feeding channel 233 are, for example, temperature-permeable and CO₂Permeability. The somatic cell culture medium storage section 223, the medium feeding path 233, and the pump 234 constitute a medium supply device.

The old medium in the 1 st amplification culture vessel 232 is sent to the waste liquid storage 228 via the waste liquid sending path 235 and the pump 236.

The somatic cells cultured in the 1 st amplification culture vessel 232 are transferred to the cell preparation site through the introduced cell liquid transfer channel 237, the pump 238, and the optional cell clump cutter 239A 2 nd expansion incubator 240 that is part of the apparatus. It is passed through a cell clump divider 239, thereby dividing the cell clumps into smaller cell clumps. In the case where the cell mass is not formed, the cell mass divider 239 may be omitted. The somatic cell culture medium is supplied from the somatic cell culture medium storage unit 223 containing the somatic cell culture medium to the 2 nd expansion culture vessel 240 through the medium feeding path 241 and the pump 242. The introduced cell liquid-feeding channel 237 and medium liquid-feeding channel 241 are, for example, temperature-permeable and CO₂Permeability. The somatic cell culture medium storage section 223, the medium feeding path 241, and the pump 242 constitute a medium replenishment device.

The old medium in the 2 nd amplification culture vessel 240 is sent to the waste liquid storage portion 228 through the waste liquid sending passage 243 and the pump 244.

The somatic cells cultured in the 2 nd expansion culture vessel 240 are sent to the solution displacer 247 via the introduced cell liquid feeding path 245 and the pump 246. The solution displacer 247 may have a structure as shown in fig. 6, for example. In the solution displacer 247 shown in FIG. 7, cells are held by the filter, and the culture medium is sent to the waste liquid storage portion 228 through the waste liquid sending passage 248 and the pump 249.

After the flow of the solution in the waste liquid feeding path 248 is stopped by stopping the driving of the pump 249, or after the waste liquid feeding path 248 is closed by a valve or the like, the frozen preservation solution is introduced from the frozen preservation solution storage unit 250 containing the frozen preservation solution to the solution displacer 247 through the liquid feeding path 251 and the pump 252. Thereby dispersing the cells in the frozen preservation solution.

The cryopreservation solution in which the somatic cells are dispersed is sent into the cryopreservation vessel 255 through a liquid sending path 253 and a pump 254 which are part of the packaging device. The frozen storage container 255 is placed in the low-temperature storage 256. Liquid nitrogen at-80 ℃ is sent from a liquid nitrogen storage 257 to a low-temperature storage 256 through a liquid sending passage 258. Thereby freezing the somatic cells in the cryopreservation vessel 255. However, freezing of somatic cells may also be liquid nitrogen independent. For example, the low-temperature storage 256 may be a compression type refrigerator, an absorption type refrigerator, a peltier type refrigerator, or the like. In addition, in the case where freezing is not required, the somatic cells may not be frozen.

A backflow prevention valve may be suitably provided in the liquid feeding passage. The liquid feeding path, the monocyte separation section 203, the monocyte purification filter 210, the factor introduction section 213, the somatic cell culture device 219, the 1 st amplification culture device 232, the 2 nd amplification culture device 240, the solution displacer 247, and the like described above are accommodated in a cassette 260 formed of resin or the like, for example, in a box shape. The case 260 is made of, for example, a heat-resistant material that can be sterilized. The case 260 is provided therein with, for example, 37 ℃ CO₂The concentration is 5% of such an environment suitable for cell culture. The liquid feeding path through which the culture medium flows is constituted of, for example, CO₂A permeable material. However, the case 260 is not limited to a box shape. For example, it may be a flexible bag. The liquid feeding path, the monocyte separation unit 203, the monocyte purification filter 210, the factor introducing unit 213, the somatic cell culture device 219, the 1 st amplification culture device 232, the 2 nd amplification culture device 240, the solution displacer 247, and the like may be divided and stored in a plurality of cartridges.

The cartridge 260 is disposed in the housing 200. The pump, blood storage unit 201, separating agent storage unit 205, factor storage unit 214, drug-containing cell culture medium storage unit 220, somatic cell culture medium storage unit 223, waste liquid storage unit 228, freezing storage container 255, low-temperature storage 256, and liquid nitrogen storage 257 are disposed inside casing 200 and outside cartridge 260.

The case 260 and the housing 200 have, for example, fitting portions that are fitted to each other. Therefore, cartridge 260 is disposed at a predetermined position in case 200. Further, in the case 200, a pump, a blood storage unit 201, a separating agent storage unit 205, a factor storage unit 214, a drug-containing cell culture medium storage unit 220, a somatic cell culture medium storage unit 223, a waste liquid storage unit 228, a freezing storage container 255, a low-temperature storage 256, and a liquid nitrogen storage 257 are disposed at predetermined positions. When the cassette 260 is disposed at a predetermined position in the casing 200, the liquid feeding path in the cassette 260 is connected to the pump, the blood storage unit 201, the separating agent storage unit 205, the factor storage unit 214, the drug-containing cell culture medium storage unit 220, the somatic cell culture medium storage unit 223, the waste liquid storage unit 228, the freezing storage container 255, the low-temperature storage 256, and the liquid nitrogen storage 257.

For example, cartridge 260 and its contents are disposable and can be discarded and replaced with a new one after freezing of the somatic cells is completed. Alternatively, when cassette 260 and its contents are reused, an identifier such as a barcode may be attached to cassette 260 to manage the number of times of use.

With the somatic cell production system according to the embodiment described above, it is possible to automatically produce somatic cells from cells before introduction without human intervention.

(other embodiments)

The present invention is described above by way of embodiments, but the description and drawings of a part of the disclosure should not be construed as limiting the present invention. Various alternative embodiments, implementations, and techniques for use will be apparent to those skilled in the art in view of this disclosure. For example, the factor-introducing device 30 may induce cells by transfection using viral vectors or plasmids such as retrovirus, lentivirus, and sendai virus, or protein transfection. Alternatively, the factor introducing device 30 may induce cells by electroporation. The pre-introduction cell feeding channel 20, the pre-introduction cell feeding channel 31, the 1 st somatic cell feeding channel 51, the amplification culture feeding channel 71, the 2 nd somatic cell feeding channel 72, and the pre-encapsulation cell channel 91 may be provided on the substrate by using a micro-fluidic technique. As described above, it is to be understood that the present invention includes various embodiments and the like not described herein.

(example 1)

12-well dishes coated with a soluble base film preparation (Matrigel, Corning) were prepared, and each well was placed with a feeder-free medium (mTeSR (registered trademark) 1, STEMCELL Technologies) containing a ROCK (Rho-associated-protein kinase/Rho-binding kinase) inhibitor (Selleck) at a concentration of 10. mu.mol/L. ROCK inhibitors inhibit cell death.

iPS cells were dispersed in a tissue/cultured Cell peeling/separating/dispersing solution (Accutase, Innovative Cell Technologies), and seeded into 12-well dishes. At 4X 10 per 1 hole⁵The transfected cells were seeded at density of individuals. In addition, the bottom area of 1 holeIs 4cm². At a rate of 2X 10 per 1 hole⁵Individual densities were seeded with untransfected control cells. Thereafter, the cells were cultured in feeder-free medium for 24 hours. At this time, the temperature was 37 ℃ and CO was₂The concentration is 5% and the oxygen concentration is 25% or less.

A transfection medium was prepared by mixing 1.25mL of Xeno-Free medium (Pluronic, STEMGENT), 0.5. mu.L of Pluronic Supplement (STEMGENT), and 2. mu.L of a solution containing recombinant B18R protein at a concentration of 100 ng/. mu.L (eBioscience). Before transfection, feeder-free medium was replaced with transfection medium for each well, and cells were cultured at 37 ℃ for 2 hours.

Green Fluorescent Protein (GFP) mRNA (TriLink) was prepared. mRNA was blocked with Anti-Reverse Cap Analog (ARCA), polyadenylated, and replaced with 5-methylcytidine and pseudouridine.

Further, 1.5mL microcentrifuge tube A and 1.5mL microcentrifuge tube B were prepared, respectively, as the number of wells.

62.5. mu.L of low serum medium (Opti-MEM (registered trademark) or Gibco) was placed in tube A, and 1.875. mu.L of a reagent for mRNA introduction (Lipofectamine MessengerMax (registered trademark) or Invitrogen) was added thereto and mixed well to prepare reaction solution 1. Thereafter, the tube A was gently tapped at room temperature for 10 minutes to mix the 1 st reaction solution.

62.5. mu.L of low serum medium (Opti-MEM (registered trademark), Gibco) was placed in tube B, and 500ng of GFP mRNA (Trilink) was added thereto and mixed well to prepare a 2 nd reaction solution.

The 2 nd reaction solution was added to the 1 st reaction solution in the tube a to prepare a mixed reaction solution, and thereafter, the tube a was gently tapped at room temperature for 5 minutes to form liposomes. Next, the mixed reaction solution was added to each well and allowed to stand at 37 ℃ overnight. Thus, 500ng of GFP mRNA was added to each well.

The cells were observed with a fluorescence microscope the next day, and as a result, coloration of the transfected cells was confirmed as shown in FIGS. 8 and 9. Further, the survival rate of the cells was confirmed as shown in FIG. 10. From this, it was found that mRNA was introduced into iPS cells using a lipofection reagent and RNA, and proteins were expressed.

(example 2)

12-well dishes coated with a soluble base film preparation (Matrigel, Corning) were prepared, and each well was placed with a feeder-free medium (mTeSR (registered trademark) 1, STEMCELL Technologies) containing a ROCK (Rho-associated-protein kinase/Rho-binding kinase) inhibitor (Selleck) at a concentration of 10. mu. mol/L. ROCK inhibitors inhibit cell death.

iPS cells were dispersed in a tissue/cultured Cell peeling/separating/dispersing solution (Accutase, Innovative Cell Technologies), and seeded into 12-well dishes. At 4X 10 per 1 hole⁵The transfected cells were seeded at density of individuals. At a rate of 2X 10 per 1 hole⁵Individual densities were seeded with untransfected control cells. Thereafter, the cells were cultured in feeder-free medium for 24 hours.

Ngn2-T2A-Puro mRNA (Trilink) and Green Fluorescent Protein (GFP) mRNA (Trilink) were prepared. mRNA was blocked with Anti-Reverse Cap Analog (ARCA), polyadenylated, and replaced with 5-methylcytidine and pseudouridine. Then, the mRNA was purified with a silica membrane, and was purified with a solution containing 1mmol/L sodium citrate of pH 6 as a solvent together with a reagent for introducing mRNA (Lipofectamine MessengerMax (registered trademark), Invitrogen). Further, 1.5mL microcentrifuge tube A and 1.5mL microcentrifuge tube B were prepared, respectively, as the number of wells.

To tube B, 62.5. mu.L of low serum medium (Opti-MEM (registered trademark), Gibco) was placed, and 500ng of Ngn2-T2A-Puro mRNA (Trilink) and 1500ng of GFP mRNA (Trilink) were added and mixed well to prepare reaction solution No. 2.

The 2 nd reaction solution was added to the 1 st reaction solution in the tube a to prepare a mixed reaction solution, and thereafter, the tube a was gently tapped at room temperature for 5 minutes to form liposomes. Next, the mixed reaction solution was added to each well and allowed to stand at 37 ℃ overnight. Thus 500ng of Ngn2mRNA and 100ng of GFPmRNA were added to each well.

After the introduction of mRNA, observation was performed on the first day, and the cell color development was confirmed as shown in FIG. 11.

On the next 2 days, mRNA-transfected cells were selected by completely replacing every 1 day the medium with neural differentiation medium (N2/DMEM/F12/NEAA, Invitrogen) containing ROCK inhibitor (Selleck) at a concentration of 10. mu. mol/L and antibiotic (puromycin) at a concentration of 1 mg/L. On day 3, the medium was replaced with neural differentiation medium (N2/DMEM/F12/NEAA, Invitrogen) containing a B18R recombinant protein-containing solution (eBioscience) at a concentration of 200 ng/mL. Thereafter, medium replacement was performed with the same medium in half the amount at a time until day 7.

On day 7, the medium was removed from the wells and washed with 1mL of PBS. Thereafter, 4% PFA was put in the reaction vessel, and the reaction was carried out at 4 ℃ for 15 minutes, followed by immobilization. Thereafter, the antibody was washed 2 times with PBS, and the primary antibody was diluted with 5% CCS and 0.1% Triton in PBS medium, and 500. mu.L of the diluted antibody was added. Primary antibody using rabbit anti-human Tuj1 antibody (BioLegend845501) and mouse anti-rat and human Ngn2 antibody (R and D Systems), rabbit anti-human Tuj1 antibody (BioLegend845501) was diluted to 1/1000 with a buffer, or mouse anti-rat and human Ngn2 antibody (R and D Systems) was diluted to 1/75 with a buffer, and DAPI was further diluted to 1/10000 with a buffer, and the reaction was performed at room temperature for 1 hour. The Tuj1 antibody is an antibody directed against β -IIITubulin.

After 1 hour of reaction at room temperature, 1mL of PBS was added to each well, and after sufficient fusion in the well, PBS was discarded. PBS was added again, discarded, and 500 μ L of permeation buffer containing secondary antibody containing 1/1000 diluted donkey anti-mouse IgG (H + L) secondary antibody Alexa Fluor (registered trademark) 555 complex (thermolisher) and 1/1000 diluted donkey anti-rabbit IgG (H + L) secondary antibody Alexa Fluor (registered trademark) 647 complex (thermolisher) were added to each permeation buffer, and reaction was performed at room temperature for 30 minutes.

After 30 minutes reaction at room temperature, the cells were washed 2 times with PBS and observed with a fluorescence microscope, and the cells that fluoresced were counted.

FIG. 12 is a photograph of a sample obtained by introducing Ngn2-T2A-Puro mRNA by lipofection, culturing the sample for 2 days with puromycin added, culturing the sample for 5 days without puromycin added, staining the sample with Tuji1, and observing the stained sample with a fluorescence microscope. FIG. 13 shows the ratio of TUJ-1 positive cells on day 7 of transfection of Ngn2-T2A-Puro mRNA with each transfection reagent by the procedure described above. From these results, it was found that nerve cells were induced.

(example 3)

12-well dishes coated with a soluble base film preparation (Matrigel, Corning) were prepared, and each well was placed with a feeder-free medium (mTeSR (registered trademark) 1, STEMCELL Technologies) containing a ROCK (Rho-associated-protein kinase/Rho-binding kinase) inhibitor (Selleck) at a concentration of 10. mu. mol/L.

iPS cells were dispersed in a tissue/cultured Cell peeling/separating/dispersing solution (Accutase, Innovative Cell Technologies), and seeded into 12-well dishes. At 4X 10 per 1 hole⁵The transfected cells were seeded at density of individuals. 1 × 10 per 1 hole⁵Individual densities were seeded with untransfected control cells. Thereafter, the cells were cultured in feeder-free medium for 24 hours. At this time, the temperature was 37 ℃ and CO was₂The concentration is 5% and the oxygen concentration is 25% or less.

A transfection medium containing B18R was prepared by mixing 1.25mL of Xeno-Free medium (Pluronic, STEMGENT), 0.5. mu.L of Pluronic Supplement (STEMGENT), and 2. mu.L of a solution containing B18R recombinant protein at a concentration of 100 ng/. mu.L (eBioscience). Then, 1.25mL of Xeno-Free medium (Pluronic, STEMGENT) and 0.5. mu.L of Pluronic Supplement (STEMGENT) were mixed to prepare a transfection medium containing no B18R.

Prior to transfection, feeder-free medium from each well was replaced with transfection medium containing B18R or transfection medium not containing B18R, and cells were cultured at 37 ℃ for 2 hours.

Ngn2-T2A-Puro mRNA (Trilink) and GFPmRNA (Trilink) were prepared. mRNA was blocked with Anti-Reverse Cap Analog (ARCA), polyadenylated, and replaced with 5-methylcytidine and pseudouridine.

To tube B, 62.5. mu.L of low serum medium (Opti-MEM (registered trademark), Gibco) was placed, and 500ng of Ngn2-T2A-Puro mRNA (Trilink) and 100ng of GFP mRNA (Trilink) were added and mixed well to prepare reaction solution 2.

The 2 nd reaction solution was added to the 1 st reaction solution in the tube a to prepare a mixed reaction solution, and thereafter, the tube a was gently tapped at room temperature for 5 minutes to form liposomes. Next, the mixed reaction solution was added to each well and allowed to stand at 37 ℃ overnight. Thus 500ng of Ngn2mRNA and 100ng of GFP mRNA were added to each well. Furthermore, as shown in FIG. 14, transfectants were prepared 1, 2, and 3 times.

On day 7, the medium was removed from the wells and washed with 1mL of PBS. Thereafter, 4% PFA was put in the reaction vessel, and the reaction was carried out at 4 ℃ for 15 minutes, followed by immobilization. Thereafter, after 2 washes with PBS, 50 μ L of primary antibody diluted with a permeation buffer containing 5% CCS and 0.1% triton x in PBS was added to each well, and the reaction was performed at room temperature for 1 hour. Primary antibody was a mouse anti-human Tuj1 antibody (BioLegend845501) diluted to 1: 1000, mouse anti-human Ngn2 antibody (R and D Systems, MAB3314-SP) was diluted to 1: 150, further comprising a ratio of 1: DAPI was added in a manner of 10,000.

After 1 hour, 1mL of PBS was added to each well, and after sufficient fusion in the well, the PBS was discarded. PBS was added again, discarded, and 500 μ L of the permeation buffer containing the secondary antibody was added to the permeation buffer, and the reaction was performed at room temperature for 30 minutes in a ratio of 1: 1000 contained the donkey anti-mouse IgG (H + L) secondary antibody Alexa Fluor (registered trademark) 555 complex (thermolisher, a-21428) and expressed in a ratio of 1: 1000 contained donkey anti-rabbit IgG (H + L) secondary antibody AlexaFluor (registered trademark) 647 complex (Thermofisiher, A31573).

The cells were washed 2 times with PBS, observed with a fluorescence microscope, and the cells that fluoresced were counted. As a result, as shown in FIG. 15, when mRNA was transfected only once, GFP could not be found at the 9 th day. On the other hand, in the case of 3-time transfection of mRNA, GFP could be found even on day 9. From this, it was found that mRNA was decomposed in cells and protein expression was transient.

As shown above, it was possible to induce neural cells several days after RNA transfection after iPS cell inoculation. Furthermore, since nerve cells can be induced in a short time, it is not necessary to include B18R protein in the culture medium, and B18R protein is generally used for suppressing cell death accompanied by immune response when RNA is inserted into cells.

Description of the symbols

2 tube

10 separating device

20 cell liquid feeding passage before introduction

21 inducing factor liquid feeding mechanism

30-factor lead-in device

31 into a cell liquid-feeding channel

40 cell manufacturing device

50 somatic cell culture device

51 somatic cell liquid feeding path

60 division mechanism

70 amplification culture device

71 amplification culture liquid feeding passage

72 somatic cell liquid feeding passage

80 division mechanism

90 somatic cell conveying mechanism

91 Pre-encapsulation cell flow channel

100 package device

101 solution displacer

102 filter

103 liquid feeding channel

104 liquid feeding channel

105 discharge flow path

106 discharge flow path

110 freezing preservation liquid feeding mechanism

200 shell

201 blood storing part

202 blood liquid feeding passage

203 monocyte separating part

204 pump

205 separating agent storage part

206 liquid feeding path

207 pump

208 monocyte liquid feeding channel

209 pump

210 monocyte purification filter

211 cell liquid feeding passage before introduction

212 pump

213 factor introducing part

Factor 214 storage unit

215 factor liquid delivery path

216 Pump

217 into cell liquid-feeding channel

218 pump

219 somatic cell culture device

220 cell culture Medium storage part

221 culture medium liquid feeding channel

222 pump

223 somatic cell culture Medium storage section

224 medium liquid feeding path

225 pump

226 waste liquid delivery passage

227 pump

228 waste liquid storage part

229 into the cell liquid-feeding channel

230 pump

231 cell mass divider

232 amplification culture device

233 medium liquid feeding path

234 pump

235 waste liquid feeding passage

236 pump

237 into the cell liquid-feeding channel

238 pump

239 cell mass divider

240 amplification culture device

241 culture medium liquid feeding passage

242 pump

243 waste liquid feeding path

244 pump

245 into the cell liquid-feeding channel

246 pump

247 solution displacer

248 waste liquid feeding channel

249 pump

250 frozen preservation solution storage part

251 liquid feeding path

252 pump

253 liquid feeding passage

254 pump

255 freezing preservation container

256 low-temperature storage

257 liquid nitrogen storage

258 liquid feeding path

259 cold storage part

260 case body

301 bag

302 bag

Claims

1. Use of a somatic cell production system for producing somatic cells other than pluripotent stem cells,

the somatic cell production system is provided with: a pre-cell introduction liquid-feeding path through which a solution containing a pre-cell to be introduced passes;

a factor introducing device connected to the pre-introduction cell liquid-feeding channel, for introducing a somatic cell-inducing factor into the pre-introduction cell to produce an inducing factor-introduced cell;

a cell preparation device for culturing the induction factor-introduced cells to prepare somatic cells; and

a cell-introducing liquid-feeding passage for feeding a solution containing the induction factor-introduced cells from the factor-introducing device to the cell-preparing apparatus.

2. The use according to claim 1, wherein the somatic cell production system further comprises a housing that houses the pre-introduction cell liquid-feeding passage, the factor-introducing device, and the cell production device.

3. The use of claim 1, wherein the somatic cells comprise differentiated cells.

4. The use of claim 1, wherein the somatic cells comprise adult stem cells.

5. The use of claim 1, wherein the somatic cells comprise neural lineage cells.

6. The use of claim 1, wherein said pre-introduction cells comprise pluripotent stem cells.

7. The use of claim 1, wherein said pre-introduced cells comprise adult stem cells.

8. The use of claim 1, wherein said pre-introduction cells comprise differentiated somatic cells.

9. The use according to any one of claims 1 to 8, wherein the cell fabrication apparatus comprises:

a somatic cell culture device for culturing the induced factor-introduced cells produced by the factor-introducing device;

an amplification culture device for performing amplification culture on the somatic cells established in the somatic cell culture device; and

a liquid feeding path for feeding a solution containing the somatic cells from the somatic cell culture apparatus to the amplification culture apparatus,

the introduced cell liquid-feeding path feeds a solution containing the induction factor-introduced cells from the factor-introducing device to the somatic cell culture device,

the somatic cell culture apparatus is provided with a 1 st medium supply device for supplying a medium to the induction factor-introduced cells,

the amplification culture apparatus is provided with a 2 nd medium replenishing apparatus for replenishing the somatic cells with a medium.

10. The use according to claim 9, wherein the somatic cell culture device is further provided with a drug replenishment device that supplies a solution containing a drug that kills cells to which the drug-resistant factor has not been introduced.

11. The use according to any one of claims 1 to 8, wherein the factor introducing device includes:

a factor-introducing part connected to the pre-introduction cell liquid-feeding channel;

a factor storage unit for storing the somatic cell-inducing factor;

a factor-delivering channel for allowing the somatic cell-inducing factor to flow from the factor-storing part to the pre-introduction cell-delivering channel or the factor-introducing part; and

a pump for causing the liquid in the factor liquid feeding passage to flow.

12. The use of claim 11, wherein the somatic cell-inducing factor is DNA, RNA, or protein.

13. The use according to claim 11, wherein the somatic cell-inducing factor is introduced into the pre-introduction cells by RNA lipofection in the factor-introducing part.

14. The use of claim 11, wherein the somatic cell-inducing factor is implanted in a carrier.

15. The use according to claim 14, wherein the vector is a Sendai virus vector.

16. The use according to claim 2, wherein the somatic cell production system is further provided with a packaging device that packages the somatic cells produced in the cell production device, and the housing accommodates the packaging device.

17. The use according to any one of claims 1 to 8, the somatic cell manufacturing system further having:

a solution displacer; and

a liquid feeding path for feeding the solution containing the somatic cells from the cell manufacturing apparatus to the solution displacer,

the solution displacer includes: a cylindrical member; and

a liquid-permeable filter disposed inside the cylindrical member,

the cylindrical member is provided with:

a somatic cell introduction hole for introducing the solution containing the somatic cells prepared in the cell preparation apparatus onto the liquid-permeable filter;

a substitution solution introduction hole for introducing a substitution solution onto the liquid-permeable filter;

a somatic cell outflow hole for allowing a substitution solution containing the somatic cells to flow out onto the liquid-permeable filter; and

and a waste liquid outflow hole for allowing the solution having permeated through the liquid permeation filter to flow out.

18. The use according to claim 17, wherein the somatic cell production system further comprises a waste liquid sending channel connected to the waste liquid outflow hole, and wherein when a solution containing the somatic cell solution is discarded, the solution in the waste liquid sending channel is allowed to flow, and when the somatic cell is dispersed in the substitution solution, the solution in the waste liquid sending channel is not allowed to flow.

19. The use according to claim 17, wherein the substitution solution is a cryopreservation solution.

20. The use according to claim 1, wherein the somatic cell production system further comprises a separation device for separating the pre-introduction cell from blood, and the solution containing the pre-introduction cell separated by the separation device passes through the pre-introduction cell liquid-sending passage.

21. The use according to claim 1, wherein the somatic cell production system further comprises a pump for sending a solution containing the inducer-introduced cells from the factor-introducing device to the introduced cell liquid-sending passage.

22. The use according to claim 9, wherein the somatic cell production system further comprises a pump that sends the solution containing the somatic cells to a liquid sending passage for sending the solution containing the somatic cells from the somatic cell culture apparatus to the amplification culture apparatus.

23. The use according to claim 17, wherein the somatic cell production system further comprises a pump that sends the solution containing the somatic cells to a liquid sending passage for sending the solution containing the somatic cells from the cell production apparatus to the solution displacer.

24. The use according to any one of claims 1 to 8, wherein the cell preparation apparatus prepares somatic cells by performing suspension culture on the inducer-introduced cells.

25. The use according to claim 9, wherein the somatic cell culture apparatus performs suspension culture on the induced factor-introduced cells produced in the factor-introducing apparatus, and the amplification culture apparatus performs amplification suspension culture on the somatic cells established in the somatic cell culture apparatus.