JP2006006125A - Method for preparing marrow mononuclear cell and apparatus for preparing cell - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a preparation method for efficiently preparing a marrow mononuclear cell by a simple operation, a cell preparation apparatus suitable for carrying out the method, a preparation method and a preparation apparatus for a marrow mononuclear cell, casting a slight burden on the collection source (e.g. human) of a marrow mononuclear cell and a method and an apparatus for simply carrying out operations from cell preparation to preparation of transplanting material. <P>SOLUTION: The method for preparing a marrow mononuclear cell comprises a filter treatment step for passing a liquid containing a marrow mononuclear cell through a porous filter and catching the marrow mononuclear cell on the filter and a cell recovery step for recovering the marrow mononuclear cell caught on the filter. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a method for preparing cells used for purposes such as regenerative medicine. Specifically, the present invention relates to a method for preparing bone marrow mononuclear cells, a preparation apparatus, and the like.

Regenerative medicine is drawing attention. Regenerative medicine has a more effective regenerative effect by transplanting stem cells collected from a living body into a defect site and a combination of stem cells collected from a living body and a scaffold material for cell proliferation. It can be classified into the method to be obtained. In either method, the most important element is considered to be the cell.
Among the cells used for regenerative medicine, a mesenchymal stem cell (MSC) is attracting particular attention. MSCs are derived from mesoderm and differentiate into cells of many tissues such as bone cells, chondrocytes, vascular endothelial cells, cardiomyocytes. In recent years, several successful cases of tissue regeneration using MSC have been reported, and some tissues are in the clinical application stage.
Although pluripotent stem cells such as embryonic stem cells are expected to be used for regenerative medicine, the reason for the high expectation for MSC is that cells that can differentiate into many tissues as described above. In addition to the characteristics, MSC can be collected from bone marrow and peripheral blood, and it is relatively easy to induce differentiation of MSC into the target tissue.
Conventionally, MSC has been generally prepared (recovered) by the following method. First, a liquid containing MSC such as bone marrow is collected from a living body, diluted with an appropriate medium, and then seeded on a culture dish. Next, after culturing for about half a day to one day, unadherent cells are washed and removed. Then, the cells remaining adhered to the culture dish are cultured under appropriate conditions, and the cells obtained by proliferation are collected as MSCs. As another MSC recovery method, a combination of centrifugation and Ficoll method is used to fractionate the mononuclear cell fraction, and then the MSC is recovered through the same dilution, seeding, and culture procedures as above. Has been. In addition, a method for separating and collecting MSC cells by FACS or MACS using an MSC cell surface marker (antibody separation method) is also used in part.
Although it is not a method specialized in the preparation of MSC, a method of preparing cells for tissue regeneration using a filter has been proposed (Patent Documents 1, 2, and 3). However, the relationship between the properties of the filter and the cell recovery rate and the utilization form of the recovered (prepared) cells have not been sufficiently studied. In addition to the techniques disclosed in the above publications, there are techniques disclosed in the following Patent Documents 4 to 6 as techniques related to the present invention.

JP 2003-319776 A JP 2002-87971 A JP 2001-161352 A JP 2003-250820 A JP 2003-52365 A JP 2003-320007 A

As described above, several methods for preparing cells for tissue regeneration are known. However, since a high recovery rate cannot be obtained or complicated operations are involved, development of a new method has been eagerly desired. In any of the above methods, a large amount of bone marrow or the like must inevitably be collected if a large amount of cells for tissue regeneration is to be prepared. On the other hand, considering the burden on the patient, the amount (number) of cells for tissue regeneration that can be prepared at one time is naturally limited. Accordingly, at present, when research and clinical application related to tissue regeneration are expected to further advance, a method (and apparatus) capable of preparing a necessary amount of cells for tissue regeneration without excessively burdening the patient is provided. It is extremely important.
In the present invention, a preparation method capable of efficiently preparing (collecting) bone marrow mononuclear cells (especially MSC) as tissue regeneration cells with a simple operation and a cell preparation apparatus suitable for carrying out the method are provided. The purpose is to provide. It is another object of the present invention to provide a preparation method and a preparation apparatus for bone marrow mononuclear cells that are less burdensome on the bone marrow mononuclear cell collection source (eg, human). Furthermore, it aims at providing the method and apparatus which can perform simply from preparation (collection) of a cell to preparation of a transplant material.

The present inventors have made extensive studies in view of the above objects. As a result, it was found that by passing a liquid containing bone marrow mononuclear cells through the porous filter, the bone marrow mononuclear cells can be captured very efficiently on the filter. In addition, when a biological sample such as bone marrow fluid is used as a cell source, it is possible to return the filtered cell source to the living body, and such a method is used for a patient who collects only necessary components. I came up with the knowledge that it would be a less burdensome (minimally invasive) procedure. In particular, if a porous filter having a pore size that enables efficient recovery of bone marrow mononuclear cells is used, the components removed by the filter are small, and the burden on the patient is extremely small.
As a result of further investigation, if a porous filter made of a bioabsorbable material is used, after capturing bone marrow mononuclear cells in the filter, the filter remains in the state of being captured by the filter without undergoing a recovery operation. It was found that bone marrow mononuclear cells can be transplanted into patients, and the process from preparation of bone marrow mononuclear cells to transplantation operation is greatly simplified.
The present invention has been completed based on the above findings, and provides the following configurations.
[1] A filtering step of passing a liquid containing bone marrow mononuclear cells through a porous filter and capturing the bone marrow mononuclear cells on the filter; and cell recovery for collecting the bone marrow mononuclear cells captured on the filter Steps,
A method for preparing bone marrow mononuclear cells.
[2] A filter treatment step of passing a liquid containing bone marrow mononuclear cells through a porous filter, capturing the bone marrow mononuclear cells on the filter, and a filter recovery step of collecting the filter containing bone marrow mononuclear cells;
A method for preparing bone marrow mononuclear cells.
[3] The preparation method according to [1] or [2], wherein the filter has a pore size of 30 μm to 500 μm.
[4] The preparation method according to any one of [1] to [3], wherein the filter is made of a biocompatible material.
[5] The preparation method according to any one of [1] to [4], wherein a step of filtering with a pre-filter having a larger pore diameter than the filter is performed before the filtering step.
[6] The preparation method according to any one of [1] to [5], wherein after the filtering step, the step of washing the filter to remove impurities is performed.
[7] The preparation method according to any one of [1] to [6], wherein all the steps are performed aseptically.
[8] The preparation method according to any one of [1] to [7], wherein the bone marrow mononuclear cells are mesenchymal stem cells.
[9] The preparation method according to any one of [1] to [8], wherein the liquid containing bone marrow mononuclear cells is bone marrow fluid.
[10] A filtering step of passing a liquid containing bone marrow mononuclear cells through a porous filter and capturing the bone marrow mononuclear cells on the filter;
A cell recovery step of recovering bone marrow mononuclear cells captured on the filter; and a cell culture step of culturing the recovered bone marrow mononuclear cells;
A method for preparing bone marrow mononuclear cells.
[11] A filtering step of passing a liquid containing bone marrow mononuclear cells through a porous filter made of a biocompatible material and capturing the bone marrow mononuclear cells on the filter;
A filter recovery step for recovering the filter;
A cell culturing step for transferring the collected filter into a culture solution and culturing bone marrow mononuclear cells while being captured by the filter; and a second filter collecting step for collecting the filter after culturing. Bone marrow mononuclear cell preparation method.
[12] A cell preparation device for preparing bone marrow mononuclear cells from a liquid containing bone marrow mononuclear cells,
An inlet for introducing the liquid, a container having an outlet for discharging the introduced liquid, and a porous filter accommodated in the container;
A cell preparation apparatus comprising:
[13] The cell preparation device according to [12], wherein the filter has a pore size of 30 μm to 500 μm.
[14] The cell preparation device according to [12], wherein the filter has a pore size of 50 μm to 200 μm.
[15] The cell preparation device according to any one of [12] to [14], wherein the filter is made of a biocompatible material.
[16] The cell preparation device according to any one of [12] to [15], further comprising suction means connected to the discharge port.
[17] The cell preparation according to any of [12] to [16], further comprising a prefilter device that is connected to the introduction port of the container and incorporates a prefilter having a pore diameter larger than that of the filter. apparatus.
[18] The cell preparation device according to any one of [12] to [17], wherein the bone marrow mononuclear cells are mesenchymal stem cells.
[19] The preparation method according to any one of [12] to [18], wherein the liquid containing bone marrow mononuclear cells is bone marrow fluid.
[20] A filter device for capturing bone marrow mononuclear cells in a liquid,
An inlet for introducing the liquid, a container having an outlet for discharging the introduced liquid, and a porous filter accommodated in the container;
A filter device comprising:
[21] The cell preparation device according to [20], wherein the filter has a pore size of 30 μm to 500 μm.
[22] The filter device according to [20], wherein the filter has a pore size of 50 μm to 200 μm.
[23] The filter device according to any one of [20] to [22], wherein the filter is made of a biocompatible material.
[24] The filter device according to any one of [20] to [23], wherein the bone marrow mononuclear cells are mesenchymal stem cells.
[25] The filter device according to any one of [20] to [24], wherein the liquid containing bone marrow mononuclear cells is bone marrow fluid.
[26] After preparing a container containing the porous filter between its inlet and outlet, the liquid containing bone marrow mononuclear cells is collected from the cell source and simultaneously through the inlet. A filtering step for introducing into the container and passing through the filter;
A liquid holding step for temporarily holding the liquid discharged from the discharge port during the first step;
A second filtering step of returning the retained liquid into the container through the outlet and passing through the filter;
A liquid reduction step for returning the liquid discharged from the inlet to the cell source during the second filter processing step; and a filter recovery step for removing the filter from the container;
A method for preparing a bone marrow mononuclear cell.
[27] The preparation method according to [26], wherein the filter has a pore size of 30 μm to 500 μm.
[28] The preparation method according to [26] or [27], wherein the filter is made of a biocompatible material.
[29] The preparation method according to any one of [26] to [28], wherein a step of filtering with a pre-filter having a larger pore size than the filter is performed before the filtering step.
[30] The preparation method according to any one of [26] to [29], wherein the filtering step to the liquid reduction step are repeated a plurality of times.
[31] The preparation method according to any one of [26] to [30], wherein all the steps are performed aseptically.
[32] The preparation method according to any one of [26] to [31], wherein the bone marrow mononuclear cells are mesenchymal stem cells.
[33] The adjustment method according to any of [26] to [32], wherein the liquid containing bone marrow mononuclear cells is bone marrow fluid.

1st aspect of this invention is related with the preparation method of a bone marrow mononuclear cell, The one aspect | mode includes the following steps.
Filtering step: A liquid containing bone marrow mononuclear cells is passed through a porous filter, and the bone marrow mononuclear cells are captured on the filter.
Cell recovery step: Bone marrow mononuclear cells captured on the filter are recovered.

In the filtering step, a liquid containing bone marrow mononuclear cells is passed through a porous filter, whereby unnecessary components are passed through the porous filter, and bone marrow mononuclear cells that are necessary components are captured on the filter. Here, the “bone marrow mononuclear cell” is used as a term of a concept including a mononuclear cell contained in the bone marrow and a cell which is the same type and contained in a body fluid or tissue other than the bone marrow. Therefore, the “liquid containing bone marrow mononuclear cells” used in the present invention is not limited to those containing cells derived from bone marrow.
On the other hand, the “liquid containing bone marrow mononuclear cells” means a biological sample in which bone marrow mononuclear cells exist as one component thereof, or a liquid prepared from the biological sample (for example, cell separation treatment and / or physiological saline). Or a liquid obtained through addition of a buffer solution or the like. Examples of biological samples here include bone marrow, periosteum, dental pulp, peripheral blood, and umbilical cord blood. The biological sample as described above is collected from an appropriate animal according to the purpose of use. The animal from which the collection is made is preferably a mammal, more preferably a rodent or a primate, and most preferably a human. The biological sample collection source (such as a tissue or an organ in which the biological sample exists) is also referred to as a “cell source” in this specification.
In particular, as the “liquid containing bone marrow mononuclear cells”, it is preferable to use a liquid containing mesenchymal stem cells, which is one of the bone marrow mononuclear cells. According to this aspect of the present invention, mesenchymal stem cells are prepared (collected). That is, a simple method for preparing mesenchymal stem cells is provided. Mesenchymal stem cells are useful for the regeneration of various tissues including bone tissue and cartilage tissue. Examples of biological samples containing mesenchymal stem cells include bone marrow, periosteum, dental pulp, peripheral blood, and umbilical cord blood. Here, “mesenchymal stem cells (hereinafter also referred to as“ MSC ”)” refers to stem cells derived from mesoderm that differentiate into cells of many tissues such as bone cells, chondrocytes, vascular endothelial cells, and cardiomyocytes. Say.

A porous filter is used in the filtering step. In particular, it is preferable to use a porous filter having a pore size in the range of 30 μm to 500 μm. According to the study by the present inventors, it is clear that a porous filter having such a pore size can efficiently capture bone marrow mononuclear cells (particularly MSCs) while suppressing the mixing of other components (for example, red blood cells). became. In order to allow more efficient capture of bone marrow mononuclear cells, a filter having a pore size of preferably 50 μm to 200 μm, more preferably 50 μm to 100 μm is used. These ranges are determined in consideration of the examination results shown in Examples described later and the size of the bone marrow mononuclear cells to be captured. When the pore size of the filter is smaller than 50 μm, unnecessary components are easily captured, so that the recovery rate of bone marrow mononuclear cells decreases. On the other hand, when the pore size of the filter is larger than 200 μm, the amount of bone marrow mononuclear cells that pass through without being captured by the filter increases, that is, the recovery rate of bone marrow mononuclear cells decreases. The pore diameter here means, in principle, the average value (average pore diameter) of the pores of the entire filter. However, the hole diameter may not be uniform over the entire filter. For example, a filter including 30% or more, preferably 50% or more, more preferably 80% or more of the total pores in the above range is also included in the filter defined above.
As described later, a non-woven filter may be used. For example, a non-woven filter having a fiber diameter of 80 μm to 200 μm in use can be used. In the case of a non-woven filter made of a hygroscopic or water-absorbing material such as collagen, a filter having the following characteristics can be used.
Fiber diameter at the time of drying: For example, 10 μm to 80 μm (preferably 30 μm to 60 μm)
Fiber diameter when wet (during use): for example, 80 μm to 200 μm (preferably 100 μm to 150 μm)

The forming material, shape and size of the porous filter used in the present invention are not particularly limited. As a material for forming the filter, for example, inorganic materials or polymer materials (synthetic polymer materials and natural polymer materials) can be used. Specifically, for example, a filter made of nylon, polyester, polyethylene, polystyrene, polypropylene, hydroxyapatite, glass, cellulose, or the like can be used.
In one embodiment of the present invention, a filter made of a biocompatible material is used. If a filter made of a biocompatible material is used, transplantation can be performed in which bone marrow mononuclear cells are captured in the filter and then bone marrow mononuclear cells are transplanted into the patient together with the filter. According to such transplantation, there is no need for an operation for recovering the bone marrow mononuclear cells captured from the filter and an operation for processing the recovered cells for transplantation (such as culture or attachment to a transplantation carrier). Transplantation by simple procedure is possible. In addition, since there is less intervention by an artificial operation, transplantation excellent in terms of safety is possible. In consideration of safety, it is preferable to use a filter made of a material having as high a biocompatibility as possible. Specifically, for example, it is preferable to use a filter made of a bioabsorbable material. Since bioabsorbable materials are absorbed and metabolized in vivo after transplantation, they are used in a wide range of applications as transplantation materials. Examples of bioabsorbable materials include synthetic polymer materials (polyglycolic acid: PGA, polylactic acid: PLA, poly-ε-caprolactone: PCL, and polymers thereof (for example, L-lactic acid-glycolic acid polymer and L-lactic acid). -ε-caprolactone copolymer), poly-p-dioxanone: PDS, etc., natural polymer materials (collagen, atelocollagen, gelatin, fibrin, polysaccharides (eg, chitin and arginine), etc.), inorganic materials (triphosphate 3) Calcium, calcium carbonate, etc.).

The filter used in the present invention can be produced by processing the above materials so as to be porous. For example, a membrane or woven filter can be produced using fiber processing technology. Moreover, a nonwoven fabric filter can be produced by a needle punch method, a melt blow method, or the like. Alternatively, a sponge-like filter can be produced using a fiber processing technique or a freeze-drying method.
In the present invention, a filter processed into a film shape (sheet shape) is typically used. However, the filter has a thickness depending on the liquid passing direction, such as a cubic shape, a rectangular parallelepiped shape, a cylindrical shape, and a spherical shape. You may use the filter processed into the shape.
A filter made of an aggregate of particles of a predetermined material and having a space (hole) between adjacent particles (adjacent particles may or may not be bonded to each other) can also be used. For example, such a filter can be prepared by filling a suitable container with particles having a predetermined particle size. On the other hand, it is also possible to use a filter (for example, a filter made of non-woven fabric) in which spaces (pores) are formed between adjacent fibers by gathering and / or overlapping fibrous materials regularly or irregularly. .

  The filtering step of the present invention may be carried out using a plurality of filters having the same or different hole diameters.

  Prior to the filtering step, it is preferable to perform pre-processing with a pre-filter for the purpose of removing dust, dirt, or agglomerated components. By carrying out such pretreatment, impurities in the liquid subjected to the filtering step are reduced, the clogging of the filter can be prevented, and efficient filtering can be performed. In addition, since the amount of contaminants attached to the filter decreases, it is expected that the amount of impurities mixed into the bone marrow mononuclear cells finally recovered will decrease. The pore diameter of the prefilter used for the pretreatment needs to be larger than the pore diameter of the filter used for the filter treatment, and is, for example, 200 μm to 1 mm.

  After the above-described filter processing step, it is preferable to perform a step of washing the filter to remove unnecessary components (contaminants) trapped or adhered to the filter. By performing such treatment, the amount of impurities mixed into the prepared bone marrow mononuclear cells can be reduced. Such a washing step can be performed using a buffer solution such as physiological saline or PBS. The washing step is performed under relatively mild conditions so as not to wash and remove bone marrow mononuclear cells captured by the filter. The cleaning conditions can be set in consideration of the type of liquid to be subjected to filter processing, the type of filter to be used, and the like. In addition, the said washing | cleaning step is implemented before the cell collection | recovery step demonstrated below.

In the cell recovery step, bone marrow mononuclear cells captured on the filter by the filtering step are recovered. The recovery step differs in the method as described below depending on the type of filter used in the filter processing step (however, the same method may be adopted even if the filter is different).
(1) When the filtering step is performed with a filter made of a non-biocompatible material In this case, in principle, it is necessary to remove the captured bone marrow mononuclear cells from the filter and collect the bone marrow mononuclear cells. Therefore, for example, as a result of the filter treatment, an appropriate recovery liquid (eg, physiological saline, buffer solution such as PBS, medium for bone marrow mononuclear cells (for example, MSC medium)) is applied to the filter in which bone marrow mononuclear cells are captured. The bone marrow mononuclear cells are collected in the collection liquid. The recovered bone marrow mononuclear cells are subjected to culture as necessary. The bone marrow mononuclear cells can also be subjected to culture without obtaining an operation of peeling the captured bone marrow mononuclear cells from the filter (that is, while being captured by the filter).
(2) When the filtering step is performed with a filter made of a biocompatible material In this case, the captured bone marrow mononuclear cells can be transplanted into the living body together with the filter. When such a use mode of bone marrow mononuclear cells is adopted, collecting the filter will collect bone marrow mononuclear cells. That is, bone marrow mononuclear cells are prepared by collecting the filter. When a biocompatible filter is used, the transplant material can be prepared without recovery of bone marrow mononuclear cells from the filter and subsequent culture (if necessary). That is, the transplant material can be quickly prepared by a very simple procedure. If a filter containing bone marrow mononuclear cells prepared in this way is used as a transplant material, the entry (new generation) of blood vessels is promoted into the transplant material after transplantation, and as a result, a good regeneration effect is obtained.
After collection of the filter (ie, collection of bone marrow mononuclear cells), the bone marrow mononuclear cells on the filter may be expanded prior to transplantation. In this case, the collected filter is transferred into a medium for bone marrow mononuclear cells and cultured under appropriate conditions.
Employment of the culture process as described above is effective when a sufficient amount of bone marrow mononuclear cells cannot be expected by filtering. Even when a biocompatible filter is used in the filter processing step, bone marrow mononuclear cells may be peeled off and collected in the same manner as when a non-biocompatible filter is used in the filter processing step.

  In one embodiment of the present invention, a bone marrow mononuclear cell prepared from a living body (cell source) is prepared (a filter is disposed between the inlet and the outlet of the container) containing the filter. A filter process is performed by introducing the liquid containing the liquid into the container through the inlet (filter process step). A liquid containing bone marrow mononuclear cells may be collected from the cell source and introduced into the container at the same time, and then discharged. The introduced liquid is discharged from the outlet of the container after passing through the filter. The discharged liquid is temporarily held (liquid holding step) and then returned to the container through the discharge port. As a result, the filtering process by the filter in the container is performed again (second filtering process step). The filtered liquid is returned to the cell source from which it was collected (liquid reduction step). After the above operation, the filter is recovered from the container (filter recovery step). When a filter made of a non-biocompatible material is used, in principle, the captured bone marrow mononuclear cells are removed from the filter and the bone marrow mononuclear cells are collected. On the other hand, when a filter made of a biocompatible material is used, bone marrow mononuclear cells are prepared by collecting the filter, and the prepared bone marrow mononuclear cells are subjected to transplantation together with the filter (cultured before transplantation). Bone marrow mononuclear cells may be proliferated). According to the preparation method comprising the above steps, only the components captured by the filter are recovered from the living body (cell source), and the burden on the living body is small. Even in the above method, it is preferable to perform pre-processing by a pre-filter before the filtering step.

  It is good also as repeating a filter collection | recovery step after repeating said filter process step-liquid reduction step several times. According to such a method, since a larger amount of liquid (liquid containing bone marrow mononuclear cells) is filtered, the amount of bone marrow mononuclear cells captured on the filter increases, and as a result, more Large quantities of bone marrow mononuclear cells can be obtained. On the other hand, since the liquid after filtering is reduced to the living body, the burden on the patient does not increase significantly. Thus, according to the above method, a large amount of bone marrow mononuclear cells can be prepared without imposing an excessive burden on the patient. The number of repetitions of the filter processing step to the liquid reduction step is not particularly limited. The number of repetitions depends on the size and pore size of the filter used, the amount of suction per time, the type of cell source, the number of bone marrow mononuclear cells in the liquid collected from the cell source, the target preparation amount of bone marrow mononuclear cells, etc. For example, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, or 9 times. Of course, the number of repetitions may be 10 or more depending on circumstances.

  In the conventional method for preparing bone marrow mononuclear cells (especially MSC), generally, a liquid containing bone marrow mononuclear cells (for example, bone marrow fluid) is collected from a living body (patient), and then the MSC is recovered by separation or the like, and the remaining liquid Was discarded. In such a method, if a large amount of bone marrow mononuclear cells (for example, MSC) is to be obtained, a large amount of bone marrow fluid must be collected and processed, which places an excessive burden on the living body. Therefore, the number of bone marrow mononuclear cells (for example, MSC) that can be prepared at one time is naturally limited. On the other hand, according to the preparation method of the present invention as described above, even if a large amount of bone marrow mononuclear cells (for example, MSC) are prepared by treating a large amount of bone marrow fluid or the like, the remaining components are returned to the living body. Therefore, the burden on the living body is small. Thus, according to the present invention, a method is provided that is minimally invasive and can prepare a large amount of bone marrow mononuclear cells at one time.

  In the preparation method of the present invention, it is preferable to perform all steps aseptically from the viewpoint of safety.

  The bone marrow mononuclear cells (or transplant material) collected or prepared using the method for preparing bone marrow mononuclear cells of the present invention can be used for tissue regeneration (reconstruction). The tissue to be regenerated is not particularly limited as long as bone marrow mononuclear cells (particularly MSC) are involved in the formation of the tissue, and suitable examples include blood vessels, bones, cartilage, teeth, periodontals, muscles, and the like. Can be cited as an organization.

  Another aspect of the present invention relates to a preparation device for bone marrow mononuclear cells. In addition, said description is used about the element and matter which are not mentioned especially. The cell preparation device of the present invention is a cell preparation device for preparing bone marrow mononuclear cells from a liquid containing bone marrow mononuclear cells, and includes a container, a filter accommodated therein, and suction means. .

The container includes an inlet for introducing a liquid containing bone marrow mononuclear cells and an outlet for discharging the introduced liquid. The container typically includes one introduction port and one discharge port, but may include either or both of these.
It is preferable that the inlet of the container has a shape and a structure that can be fitted with an injection needle or the like so that the liquid can be introduced into the container through an easy procedure. From the viewpoint of operability, it is particularly preferable to employ an introduction port so that an injection needle or the like can be attached without using a special device or the like when using (when preparing cells).

  A porous filter (see the above description) is accommodated in the container. The filter is housed between the inlet and the outlet of the container (position between the two) so that the liquid introduced from the inlet once passes through the filter and is discharged from the outlet. It is preferred that the inlet and outlet are completely separated by the filter so that all of the introduced liquid passes through the filter before draining, thereby allowing efficient capture of bone marrow mononuclear cells.

  The cell preparation device of the present invention may further include a suction means. The suction means is connected to the outlet of the container. The liquid is introduced into the container through the inlet using the suction force of the suction means. The suction means is not particularly limited as long as it can be connected to the discharge port of the container and can introduce the liquid as described above. For example, a pump or a piston can be adopted as the suction means. Specifically, for example, a syringe can be used as the suction means.

It is preferable that the suction means has a discharge capability in the container direction in addition to a suction capability from the container. According to such a suction means, the liquid introduced into the container through the introduction port by the suction ability, passed through the filter, and discharged through the discharge port is again introduced into the container through the discharge port. It becomes possible to do. The liquid introduced into the container passes through the filter and is then discharged through the inlet. As a result of a series of suction and discharge operations by the suction means as described above, the liquid to be processed (liquid containing bone marrow mononuclear cells) passes through the filter twice and is discharged outside the apparatus (outside the container). Here, if the above operation is performed with the inlet side of the container connected to the cell source, the liquid containing bone marrow mononuclear cells collected from the cell source is reduced to the cell source after filtering. . Taking bone marrow mononuclear cells from bone marrow as an example, the bone marrow fluid collected from a patient is collected in the same patient after the bone marrow mononuclear cells in the bone marrow are captured by the filter by the above-described collection operation by the suction means. Will be returned. Therefore, only the component captured by the filter is taken out from the patient, and the burden on the patient is small. In addition, the burden on the patient does not increase significantly even when repeated collection operations are performed. In this way, a method of preparing bone marrow mononuclear cells with very little burden on the patient has been implemented by adopting suction means that also has the ability to discharge into the container in addition to the ability to suck from the container. A possible cell preparation device. Here, “patient” means a subject from which bone marrow mononuclear cells are collected (that is, a living body including a cell source), and is not necessarily the same as a transplant subject of recovered bone marrow mononuclear cells.
Hereinafter, the present invention will be described in more detail with reference to the drawings.

A cell preparation apparatus 1 according to an embodiment of the present invention is shown in FIG. The cell preparation device 1 includes a filter unit 10, an injection needle 20 (for example, 16 to 24 gauge), a syringe (no needle) 30, and a connecting member 40. The filter unit 10 includes a filter container 11 having an introduction port 12 and a discharge port 13 and a cell trapping filter 14 accommodated in the filter container 11. The filter container 11 is hollow, and an introduction port 12 and a discharge port 13 are formed so as to protrude from the center of the upper surface and the lower surface. The introduction port 12 and the discharge port 13 each have a cylindrical shape, and the inner space of both is communicated via the inner space of the filter container main body. The filter container main body can be disassembled into two parts at the top and bottom and can be connected. In this embodiment, such a free disassembly and connection can be achieved by a screw type connection method. The material of the filter container 11 is polystyrene.
In use, the cell-capturing filter 14 is housed in the filter container body. The cell trapping filter 14 is housed in the center of the filter container main body so as to divide the filter container main body into two spaces (the cell capture filter 14 is introduced into the inlet 11 and the outlet of the filter container 11). 12). In this example, a porous filter made of a non-woven fabric made of collagen having an average fiber diameter of about 30 μm to about 60 μm when dried and an average fiber diameter of about 100 μm to about 150 μm when wet was used as the filter 14 for capturing cells.
The injection needle 20 is connected to the inlet 12 of the filter container 11 via the connecting portion 40. An injection needle with an appropriate inner diameter is employed according to the object (cell source) of the cell preparation device 1. In this example, an 18 gauge needle was used to prepare bone marrow mononuclear cells from bone marrow.
A syringe 30 is connected to the discharge port 13 of the filter container 11. In this example, a 50 ml syringe was used.

Next, a cell preparation method using the cell preparation apparatus 1 will be described with reference to the flowchart of FIG. 4 by taking preparation of bone marrow mononuclear cells from bone marrow as an example. First, the cell preparation device 1 is prepared aseptically. Next, after performing an incision as necessary, the tip of the injection needle 20 of the cell preparation device 1 is inserted into the bone marrow which is a cell source. Subsequently, the plunger 31 of the syringe 30 is pulled in this state. The resulting suction force causes bone marrow fluid to pass through the injection needle 20 and then into the filter container 11 via its inlet 12. The introduced bone marrow fluid is filtered by the cell capture filter 14 in the filter container 11 (filtering step: S1). By this filtration, bone marrow mononuclear cells are captured on the cell capturing filter 14. The filtered bone marrow is discharged from the filter container 11 through the discharge port 13 and held in the syringe 30. Next, in order to collect the bone marrow mononuclear cells captured on the cell capturing filter 14, the cell capturing filter 14 is taken out from the filter container 11. The removed cell-capturing filter 14 is washed with physiological saline or an appropriate buffer to recover bone marrow mononuclear cells (cell recovery step: S2). The recovered bone marrow mononuclear cells are subjected to culture as necessary (culture step: S3).
Components (contaminants) other than bone marrow mononuclear cells captured (or adsorbed) by the cell-capturing filter 14 after the above filtering step are removed, and the purity of the bone marrow mononuclear cells finally recovered is determined. For the purpose of enhancing, it is preferable to perform a step (contaminant removing step: S4) of washing the cell capturing filter 14 with an appropriate liquid between the filtering step and the cell recovery step. This step is performed as follows, for example. First, the syringe is removed from the filter container 11 after the filtering step. Subsequently, another syringe holding the cleaning liquid is connected to the outlet 13 of the filter container 11. Then, the liquid in the syringe is injected into the filter container 11 through the discharge port 13. The injected liquid passes through the cell capturing filter 14 and is then discharged out of the container through the inlet 12 of the filter container 11. As described above, the cleaning effect is obtained by passing the cleaning liquid through the cell trapping filter 14, and impurities on the cell trapping filter 14 can be removed.

Next, another example of a method for recovering bone marrow mononuclear cells using the cell preparation device 1 will be shown (see the flowchart in FIG. 5). First, the tip of the injection needle 20 of the cell preparation device 1 is inserted into the bone marrow, which is a cell source. Subsequently, the bone marrow fluid is taken in through the injection needle 20 by pulling the plunger 31 of the syringe 30 and is subjected to a filter process (filter process step: S11). At this stage, the tip of the injection needle 20 is not taken out from the bone marrow, and the tip of the injection needle 20 is maintained in the bone marrow fluid until the filter recovery step described below is performed.
As a result of performing the filtering step, the bone marrow after filtering is held in the syringe 30 (liquid holding step: S12). Next, by pushing the plunger 31 of the syringe 30, the bone marrow held in the syringe 30 is returned to the filter container 11 in which the cell capturing filter is accommodated. Thereby, the bone marrow fluid is filtered again (second filter processing step: S13). The filtered bone marrow fluid is reduced into the bone marrow through the injection needle 20 (liquid reduction step: S14).
The liquid reduction step is repeated a predetermined number of times from the above filtering step. After completion of the predetermined number of times, the cell trapping filter is taken out (filter recovery step). The taken-out cell capture filter can be used as it is as a graft (graft for bone marrow mononuclear cell transplantation). Alternatively, the taken-out cell capture filter may be transferred into an appropriate medium, and the bone marrow mononuclear cells captured on the filter may be cultured. In this case, the cell trapping filter is used as a scaffold for bone marrow mononuclear cells. Alternatively, after recovering bone marrow mononuclear cells from the taken-out cell capturing filter by means such as washing, the recovered bone marrow mononuclear cells can be used for culture or used for transplantation. Here, the predetermined number of times is usually a number of times (for example, 1 to 7 times) expected to capture a target amount of bone marrow mononuclear cells on the cell capturing filter.
According to the above method, there is substantially no component collected from the living body except for the component captured by the cell capturing filter. Therefore, the burden on the living body is greatly reduced. In addition, since the bone marrow fluid that has been filtered once is filtered again, an improvement in the capture rate of bone marrow mononuclear cells on the cell capture filter can be expected. Further, by repeatedly performing the liquid reduction step from the filtering step, a large amount of bone marrow fluid is subjected to the filtering process, and more bone marrow mononuclear cells can be captured on the filter. On the other hand, bone marrow mononuclear cells prepared by the above method are captured on a filter made of a bioabsorbable material and can be used as an implant as it is (that is, with the filter). Thus, in the above method, the recovery of bone marrow mononuclear cells and the preparation of a graft are performed at the same time, and a transplant material containing bone marrow mononuclear cells can be easily and rapidly prepared.

Next, the structure and usage of the cell preparation device 2 which is another embodiment will be described. In the drawings used for the following description, the same members as those of the cell preparation device 1 are denoted by the same reference numerals, and the description thereof is omitted. As shown in FIG. 2, the cell preparation device 2 includes a filter unit 15 and a prefilter unit 17. The prefilter unit 17 is attached between the injection needle 20 and the filter unit 15.
In the filter unit 15, a porous filter (cell capture filter 16) made of Teflon (registered trademark: tetrafluoroethylene) having a pore diameter of about 200 μm is housed. On the other hand, a Teflon (registered trademark) porous filter (prefilter 18) having a pore diameter of about 500 μm is accommodated in the prefilter portion 17. Other configurations are the same as those of the cell preparation device 1. In such a cell preparation apparatus 2, bone marrow mononuclear cells are collected by the following procedure (see the flowchart in FIG. 6). In the following, a method for recovering bone marrow mononuclear cells from bone marrow will be shown as an example.
First, the cell preparation device 2 is prepared aseptically. Next, after performing an incision as necessary, the tip of the injection needle 20 of the cell preparation device 2 is inserted into the bone marrow which is a cell source. Subsequently, the plunger 31 of the syringe 30 is pulled in this state. The resulting suction force causes bone marrow fluid to pass through the injection needle 20 and then into the prefilter portion 17. The introduced bone marrow fluid is filtered from the prefilter 18 and then discharged from the outlet of the prefilter unit 17 (prefiltering step: S0). As a result of the filtering action by the prefilter 17, bone fragments and dust in the bone marrow fluid are removed. The bone marrow fluid discharged from the prefilter unit 17 is subsequently introduced into the filter unit 15. Thereafter, as in the case of the cell preparation device 1, the filter processing step (S1), the cell recovery step (S2), and the culture step (S3) are sequentially performed.
As described above, when the cell preparation device 2 is used, pre-processing by the pre-filter 18 is performed before the filtering step by the cell capturing filter 16. By this pretreatment, bone marrow fluid from which large-sized contaminants such as bone fragments have been removed in advance is used for the filtering step. As a result, in the filtering step, a better filtration effect is achieved, and the capture rate of bone marrow mononuclear cells is improved and the amount of contaminants is reduced.
As in the case of the cell preparation apparatus 1, a contaminant removal step (S4) is performed between the filtering step (S1) and the cell recovery step (S2), and the purity of the recovered bone marrow mononuclear cells is determined. It is preferable to increase.

  In particular, when a filter made of a bioabsorbable material (for example, a non-woven collagen filter) is used as the filter 16, bone marrow mononuclear cells can be prepared by the following procedure (see the flowchart in FIG. 7). First, a cell preparation device is prepared aseptically. Next, after performing an incision as necessary, the tip of the injection needle 20 of the cell preparation device is inserted into the bone marrow which is a cell source. Subsequently, the plunger 31 of the syringe 30 is pulled in this state. By this operation, the bone marrow fluid is introduced into the prefilter part 17 through the injection needle 20, and is subjected to a filtering action by the prefilter 18 (prefiltering step: S10). After this pretreatment, a liquid holding step to a filter number step are performed in the same procedure as described above (S11 to S16), and bone marrow mononuclear cells captured on a cell capturing filter made of a bioabsorbable material are obtained. . According to the method as described above, a better filtering action is achieved in the filtering step, and the capture rate of bone marrow mononuclear cells is improved and the amount of contaminants is reduced. Further, as in the case of the above method, many bone marrow mononuclear cells can be collected without imposing an excessive burden on the living body, and a simple and rapid preparation of a transplant material containing bone marrow mononuclear cells can be achieved. It becomes possible.

Next, the configuration and usage of a cell preparation device 3 that is still another embodiment will be described. In the drawings used for the following description, the same members as those of the cell preparation device 2 are denoted by the same reference numerals, and the description thereof is omitted. As shown in FIG. 3, the cell preparation device 3 includes a filter unit 15 and two pre-filter units 17a and 17b. One end of the pre-filter part 17a is connected to the container 50. Moreover, each filter part is connected so that the pre filter part 17a, the pre filter part 17b, and the filter part 15 may be located in order from the container 50 side. Each of the prefilter portions 17a and 17b accommodates a Teflon (registered trademark) porous filter (prefilters 18a and 18b) having a pore diameter of about 500 μm. On the other hand, the filter unit 15 accommodates a porous filter (cell capture filter 16) made of Teflon (registered trademark: tetrafluoroethylene) having a pore diameter of about 200 μm.
In the cell preparation device 3, a cell source (for example, collected bone marrow fluid) is placed in a container 50, and the cell source is sequentially passed through the prefilter unit 17 a, the prefilter unit 17 b, and the filter unit 15 using the action of gravity. Hereinafter, a case where bone marrow fluid is used as a cell source will be described as an example of a method for recovering bone marrow mononuclear cells by the cell preparation device 3.
First, the cell preparation device 3 is prepared aseptically. On the other hand, bone marrow fluid is collected from the iliac using a separately prepared syringe or the like. Next, the collected bone marrow fluid is transferred to the container 50 and then left for a while. As a result, the bone marrow fluid first passes through the prefilter parts 17a and 17b. At this time, the filtering action by the pre-filters 18a and 18b is exerted, and bone fragments and dust in the bone marrow fluid are removed. The bone marrow fluid discharged from the prefilter unit 17b is subsequently introduced into the filter unit 15. The introduced bone marrow fluid is filtered by the cell capturing filter 16 in the filter unit 15. By this filtration, bone marrow mononuclear cells are captured on the cell capturing filter 16. The bone marrow after filtration is discharged from the open end of the filter unit 15. Next, the cell capture filter 16 is collected and washed with physiological saline or an appropriate buffer to collect bone marrow mononuclear cells. The recovered bone marrow mononuclear cells are subjected to culture as necessary.
As described above, when the cell preparation device 3 is used, pre-processing by the pre-filters 18 a and 18 b is performed before the filtering step by the cell capturing filter 16. By this pretreatment, bone marrow fluid from which large-sized contaminants such as bone fragments have been removed in advance is subjected to a filtering step by the cell capturing filter 16. As a result, in the filtering step, a better filtration effect is achieved, and the capture rate of bone marrow mononuclear cells is improved and the amount of contaminants is reduced.

An attempt was made to prepare bone marrow mononuclear cells from bone marrow using the cell preparation apparatus 1 described above. The experimental method is described below. First, a collagen non-woven fabric (porous filter) was packaged in a filter container, and a syringe was attached to obtain a cell preparation device 1. Using the cell preparation device 1, the bone marrow fluid was aspirated from the rabbit iliac and then returned again. Heparinized collagen non-woven fabric was easily clogged to prevent blood from solidifying, and the operation of returning the sucked liquid was limited to 2 to 3 times.
After the above operation, the collagen nonwoven fabric was collected. Subsequently, the cells captured by the collagen nonwoven fabric were collected by washing the collagen nonwoven fabric with a culture solution. The collected cells were subjected to culture, and the number of colonies and the number of cells grown as a result were counted. In addition, an osteoinduction medium was added after culturing for a predetermined time, and ALP activity was measured. On the other hand, mononuclear cells separated from 1-2 ml of bone marrow fluid were cultured as a comparison target (control). As a result, the number of colonies and the number of cells grown were counted. In addition, an osteoinduction medium was added in the same manner as described above, and ALP activity was measured.
From each measurement result (not shown), MSC-like cells could be cultured from the cells trapped by the collagen nonwoven fabric to the same extent as the control. In addition, as in the control, an increase in ALP activity was observed, confirming that MSC-like cells were proliferating. The number of colonies formed (colony forming ability) when cells trapped by the collagen nonwoven fabric were used was equivalent to the number of colonies obtained from 1-2 ml of bone marrow fluid as a control. From this result, it can be said that the above method is a very useful cell separation method.

An attempt was made to prepare bone marrow mononuclear cells from human bone marrow using the cell preparation apparatus 3 described above. The experimental method is described below. A Teflon (registered trademark: tetrafluoroethylene) porous filter having an average pore diameter of 200 μm and a Teflon (registered trademark) porous filter having an average pore diameter of 500 μm were prepared, and each was packaged in a filter container. Then, each filter container was connected, the container for accommodating bone marrow fluid temporarily was attached, and it was set as the cell preparation apparatus 3. FIG. Meanwhile, about 400 ml of bone marrow fluid was collected from human iliac bone. The collected bone marrow fluid was transferred to the container 50 of the cell preparation device 3 and then left for a while. Next, after confirming that the entire amount of bone marrow fluid was discharged from the open end of the filter unit 15, each filter (16, 18a, and 18b) was recovered. Subsequently, the cells captured by each filter were collected by washing each collected filter with a culture solution. The collected cells were each subjected to culture, and the number of colonies and the number of cells grown as a result were counted. In addition, an osteoinduction medium was added after culturing for a predetermined time, and ALP activity was measured. On the other hand, mononuclear cells isolated from 1 ml of bone marrow fluid were cultured as a comparison target (control). As a result, the number of colonies of the proliferated cells was counted. In addition, an osteoinduction medium was added in the same manner as described above, and ALP activity was measured.
The number of colonies formed on the third day of culture is shown in the graph of FIG. Further, the number of cells on the 10th day of culture is shown in the graph of FIG. On the other hand, ALP activity is shown in the graph of FIG. In FIG. 9, SC represents the ALP activity of the group that did not induce bone (group cultured with MSC as it was), and ind represents the ALP activity of the group that induced bone induction in the bone induction medium.
As shown in FIG. 8, it can be seen that in both the filter having a pore diameter of 500 μm and the filter having a pore diameter of 200 μm, cells can be recovered with an efficiency equal to or higher than that of the control. In addition, a higher cell recovery rate is obtained with a filter having a pore diameter of 200 μm than with a filter having a pore diameter of 500 μm. On the other hand, the recovered cells showed an increase in ALP activity, confirming the proliferation of MSC-like cells (FIG. 9).
From the above results, (1) filter processing using a porous filter is effective for recovery of MSC, and (2) both a filter with an average pore diameter of 500 μm and a filter with an average pore diameter of 200 μm are good. It was found that a cell recovery rate was obtained, and (3) a filter with an average pore size of 200 μm was superior to a filter with an average pore size of 500 μm.

Regeneration of cartilage tissue using cells collected from bone marrow fluid was attempted by the following procedure. The cell preparation apparatus 1 described above was used for cell collection. First, a cell preparation device 1 was obtained by packaging a PGA (polyglycolic acid) nonwoven fabric (porous filter) in a filter container and attaching a syringe. Using the cell preparation apparatus 1, bone marrow fluid (about 3 ml to 5 ml) was aspirated from the bone marrow cavity of the rabbit joint. Thereafter, the PGA nonwoven fabric was collected. Using the thus obtained cell-containing PGA nonwoven fabric, the following procedure was performed.
Anesthesia: Ketamine (Brand name Ketaral, Sankyo Ale Yakuhin Co., Ltd.) 35mg / kg im + Xylazine (Brand name Seractal, Bayer) 5mg / kg im
Anesthesia time: 25-40 minutes, Awakening time: 60-120 minutes <Surgery procedure>
1) A rabbit (Japanese white rabbit, age 10-20) was anesthetized by the above method.
2) Hair was removed from the thigh to the lower leg using a shaver and hair removal cream.
3) An approach according to the anterior knee approach was used (longitudinal incision in front of knee, inside of patella separated and invading into joint).
4) A cartilage defect of about 5mm (up to the subchondral bone) was made using Ruell-file.
5) The cell-containing PGA non-woven fabric prepared by the above method was filled in the cartilage defect (PGA cell (+) group). In that case, it decided to fix with an absorptive thread | yarn. In addition, as a control (control), a group (PGA cell (−) group) transplanted with a non-bone marrow fluid non-bone marrow fluid and a group (simple defect group) in which nothing was transplanted in the defect part were provided.
5) A sheet of HA + cellulose (sepra film, registered trademark, manufactured by Genzyme Japan Co., Ltd.) was placed in the joint space to prevent adhesion. The surgery was completed with closure, gauze dressing and gypsum fixation.

After the above operation, the transplanted tissue was removed after a certain period of time and evaluated macroscopically and histologically. The results are shown in FIGS. FIG. 10 is a macroscopic observation of the state of the transplanted part 4 months after the transplantation of the PGA cell (+) group (left) and a HE-stained image of the transplanted part section (right), and FIG. 11 shows the PGA cell. It is the same figure (left: macroscopic observation image, right: HE stained image) of (-). On the other hand, FIG. 12 is a macroscopic observation of the state of the defect formation part two months after the defect formation of the simple defect group (left) and an HE-stained image of the defect formation part section (right).
As shown in FIGS. 10 to 12, in the PGA cell (+) group, better cartilage formation (regeneration) is recognized as compared to the PGA cell (−) group. There is also a clear difference in comparison with the simple defect group. From the above results, it was shown that the above method using a PGA nonwoven fabric is effective for regeneration of cartilage tissue. That is, it becomes clear that cells for regeneration of cartilage (that is, MSC) can be captured by the PGA nonwoven fabric, and the cell-containing PGA nonwoven fabric thus obtained can be effectively used as a transplant material for regeneration of cartilage tissue as it is. There was found.

  The bone marrow mononuclear cell preparation method or preparation apparatus of the present invention is used for recovering bone marrow mononuclear cells from bone marrow or peripheral blood. The present invention is particularly useful for recovering mesenchymal stem cells. The recovered bone marrow mononuclear cells can be used particularly in the field of regenerative medicine, such as bone tissue, cartilage tissue, adipose tissue, periodontal tissue, blood vessel, ligament, tendon, muscle, or myocardial reconstruction.

The present invention is not limited to the description of the embodiments and examples of the invention described above. Various modifications may be included in the present invention as long as those skilled in the art can easily conceive without departing from the description of the scope of claims.
The contents of papers, published patent gazettes, patent gazettes, and the like specified in this specification are incorporated by reference in their entirety.

FIG. 1 is a diagram showing a cell preparation apparatus 1 according to an embodiment of the present invention. FIG. 2 is a diagram showing a cell preparation device 2 which is another embodiment. FIG. 3 is a diagram showing a cell preparation device 3 which is another embodiment. FIG. 4 is a flowchart of a method for preparing bone marrow mononuclear cells using the cell preparation device 1. FIG. 5 is a flowchart showing another example of a method for recovering bone marrow mononuclear cells using the cell preparation device 1. FIG. 6 is a flowchart of a method for preparing bone marrow mononuclear cells using the cell preparation device 2. FIG. 7 is a flowchart of a method for preparing bone marrow mononuclear cells using a filter for capturing cells made of a bioabsorbable material and performing prefiltering. FIG. 8 shows the cell recovery rate of each filter when MSC was recovered from human bone marrow using the cell preparation device 3. (A) is a graph showing the number of colonies formed on the third day of culture, and (B) is a graph showing the number of cells on the seventh day of culture. FIG. 9 is a graph showing ALP activity during bone induction of the recovered MSC when MSC is recovered from human bone marrow fluid using the cell preparation device 3. FIG. 10 is a diagram showing the results of a transplantation experiment using a transplant material obtained using the cell preparation device 1, and visually observing the state of the transplanted part 4 months after the transplantation of the PGA cell (+) group. 2 is a HE-stained image (right) of a transplanted part (left) and a transplanted section. FIG. 11 is a diagram showing the results (comparison control) of the transplantation experiment using the transplant material obtained using the cell preparation device 1, and shows the state of the transplanted part 4 months after the transplantation of the PGA cell (−) group. A macroscopic observation (left) and a HE-stained image (right) of the transplanted section. FIG. 12 is a diagram showing the results (comparison control) of the transplantation experiment using the transplant material obtained using the cell preparation device 1, and the state of the defect formation part two months after the defect formation of the simple defect group is visually observed. It is what was observed automatically (left), and the HE dyeing | staining image (right) of a defect formation part slice.

Explanation of symbols

1, 2, 3 Cell preparation device 10 Filter unit 11 Filter container 14 Cell trapping filter (collagen nonwoven fabric)
16 Cell capture filter (porous filter with an average pore size of 200μm)
17 17a 17b Pre-filter part 18 Pre-filter 18a, 18b Porous filter 20 with average pore diameter of 500 μm Injection needle 30 Syringe 31 Plunger 40 Connecting member

Claims (19)

Filtering step of passing a liquid containing bone marrow mononuclear cells through a porous filter, capturing bone marrow mononuclear cells on the filter, and cell recovery step of collecting bone marrow mononuclear cells captured on the filter,
A method for preparing bone marrow mononuclear cells. A filtering step of passing a liquid containing bone marrow mononuclear cells through a porous filter and capturing the bone marrow mononuclear cells on the filter; and a filter recovery step of collecting the filter containing bone marrow mononuclear cells;
A method for preparing bone marrow mononuclear cells.   The preparation method according to claim 1 or 2, wherein the filter has a pore size of 30 µm to 500 µm.   The preparation method according to claim 1, wherein the filter is made of a biocompatible material.   The preparation method according to any one of claims 1 to 4, wherein a step of filtering with a pre-filter having a larger pore diameter than the filter is performed before the filtering step.   The preparation method according to any one of claims 1 to 5, wherein after the filtering step, a step of washing the filter to remove impurities is performed.   The preparation method according to claim 1, wherein all the steps are performed aseptically.   The preparation method according to any one of claims 1 to 7, wherein the bone marrow mononuclear cells are mesenchymal stem cells.   The preparation method according to claim 1, wherein the liquid containing bone marrow mononuclear cells is bone marrow fluid. A filtering step of passing a liquid containing bone marrow mononuclear cells through a porous filter and capturing bone marrow mononuclear cells on the filter;
A cell recovery step of recovering bone marrow mononuclear cells captured on the filter; and a cell culture step of culturing the recovered bone marrow mononuclear cells;
A method for preparing bone marrow mononuclear cells. A filtering step of passing a liquid containing bone marrow mononuclear cells through a porous filter made of a biocompatible material and capturing bone marrow mononuclear cells on the filter;
A filter recovery step for recovering the filter;
A cell culturing step for transferring the collected filter into a culture solution and culturing bone marrow mononuclear cells while being captured by the filter; and a second filter collecting step for collecting the filter after culturing. Bone marrow mononuclear cell preparation method. A cell preparation device for preparing bone marrow mononuclear cells from a liquid containing bone marrow mononuclear cells,
An inlet for introducing the liquid, a container having an outlet for discharging the introduced liquid, and a porous filter accommodated in the container;
A cell preparation apparatus comprising:   The cell preparation device according to claim 12, wherein the filter has a pore size of 30 μm to 500 μm.   The cell preparation device according to claim 12, wherein the filter has a pore size of 50 μm to 200 μm.   The cell preparation device according to any one of claims 12 to 14, wherein the filter is made of a biocompatible material.   The cell preparation device according to any one of claims 12 to 15, further comprising suction means connected to the discharge port.   The cell preparation device according to any one of claims 12 to 16, further comprising a prefilter device that is connected to the introduction port of the container and incorporates a prefilter having a pore diameter larger than that of the filter.   The cell preparation device according to any one of claims 12 to 17, wherein the bone marrow mononuclear cells are mesenchymal stem cells.   The preparation method according to any one of claims 12 to 18, wherein the liquid containing bone marrow mononuclear cells is bone marrow fluid.