CN106801038A - A kind of utilization Three-dimensional cell culture system promotes the cell culture processes of umbilical cord blood hematopoietic stem cell fast and stable propagation - Google Patents

Abstract

The present invention proposes the cell culture processes that a kind of utilization Three-dimensional cell culture system promotes umbilical cord blood hematopoietic stem cell fast and stable propagation, comprises the following steps：1) cell for preparing and being used as stroma cell when training altogether is extracted, and is identified with flow cytometry；2) material such as the microcarrier for being used during treatment dimensional culture or other carriers, it is standby；3) by inoculated stromal cell to microcarrier, cultivated in three dimensional culture system；4) after after stromal cell proliferation to covering micro-carrier surface more than 80%, cell is processed, destroys mitosis；5) the candidate stem cell culture medium of homogeneous is prepared；6) PBMC is separated from Cord blood, CD34+ cells is sub-elected after obtaining PBMC, identified with flow cytometry；7) will sort the CD34+ cells for obtaining adds the candidate stem cell culture medium of homogeneous to be cultivated with treated stroma cell in three dimensional culture system.The present invention is established combine with stroma cell, cytokine profiles using Three-dimensional cell culture system and promotes that candidate stem cell is quick within the regular hour, the stable culture systems bred.Theoretical foundation and technology platform are provided clinically to obtain high-quality candidate stem cell.

Description

A cell culture method that uses a three-dimensional cell culture system to promote the rapid and stable proliferation of umbilical cord blood hematopoietic stem cells Law

technical field

The invention belongs to the field of biotechnology, and relates to a cell culture method for promoting rapid and stable proliferation of umbilical cord blood hematopoietic stem cells by using a three-dimensional cell culture system

Background technique

Hematopoietic stem cell transplantation is an effective method for the clinical treatment of severe trauma, malignant blood diseases, severe aplastic anemia, immunodeficiency syndrome, thalassemia and other diseases. Since the end of the 1980s, the world's first umbilical cord blood hematopoietic stem cell transplantation successfully treated thalassemia patients and the gradual establishment and improvement of cord blood banks around the world. It is a good supplement to bone marrow transplantation and has been increasingly used in the treatment of various blood system diseases. At present, more than tens of thousands of children and adults have received UCBT worldwide. However, in clinical practice, cord blood transplantation also encountered obstacles. Due to the small absolute number of cord blood hematopoietic stem cells, cord blood transplantation can only be used in children and low-weight adult patients, which is difficult to meet the needs of adult transplantation. In addition, the recovery of neutrophils and platelets after umbilical cord blood transplantation is slower than that of bone marrow and mobilized peripheral blood. Thus limiting its clinical application.

In order to expand the scope of umbilical cord blood transplantation and accelerate the recovery of neutrophils and platelets after transplantation, it is necessary to expand the hematopoietic stem/progenitor cells of umbilical cord blood in vitro, and generate enough progenitor cells for transplantation to adults through in vitro expansion methods , and the expanded cord blood hematopoietic cells in vitro should contain hematopoietic stem cells and early hematopoietic progenitor cells with long-term reconstitution hematopoietic activity. At present, the conventional method for in vitro expansion of hematopoietic stem and progenitor cells is mostly two-dimensional culture, which is to culture hematopoietic stem and progenitor cells in a single suspension, relying on the combined action of high doses of various cytokines, or to combine hematopoietic stem cells with exogenous Cytokines are mixed with umbilical cord mesenchymal stem cells, placental mesenchymal stem cells, AGM area cells and other feeder cells related to hematopoiesis. However, this in vitro expansion method has obvious limitations. ① Cells can only form a monolayer in the horizontal line , it is difficult to approach the three-dimensional environment in the body. ② Cells cultured in a two-dimensional environment are different from those in the three-dimensional space system under physiological conditions in the contact between cells and the interaction between cells and substrates. ③ The limitation of nutrient intake and the change of cell metabolism lead to the limitation of cell activity. Therefore, cells expanded in such culture systems are unable to reconstitute hematopoiesis in the long term.

HSC can well maintain its self-renewal and multi-directional differentiation potential in vivo, which depends on the microenvironment in vivo, including cytokines and hematopoietic growth factors, interaction with supporting cell types and extracellular matrix molecules, three-dimensional spatial configuration, etc. . In this research, hematopoietic stem cells, exogenous cytokines and feeder cells are co-cultured in a three-dimensional culture system, which can more fully simulate the hematopoietic microenvironment in vivo. The experimental results show that the culture results of this system show strong in vitro expansion ability, and has the potential to maintain HSC self-renewal and multilineage differentiation. In view of this, the present invention can rapidly and stably amplify umbilical cord blood hematopoietic stem cells during in vitro culture, and obtain high-quality functional hematopoietic stem cells, which is of great clinical significance.

Contents of the invention

The present invention proposes a cell culture method using a three-dimensional cell culture system to promote the rapid and stable proliferation of cord blood hematopoietic stem cells, which solves the technical bottleneck of insufficient hematopoietic stem cells in the hematopoietic stem cell transplantation treatment of the prior art in clinical practice.

The technical solution of the present invention is achieved in the following way: a cell culture method for promoting rapid and stable proliferation of umbilical cord blood hematopoietic stem cells by using a three-dimensional cell culture system, comprising the following steps:

1) extract and prepare the cells used as stromal cells during co-cultivation, and identify them by flow cytometry; 2) process materials such as microcarriers or other carriers used in the three-dimensional culture process, and reserve them; 3) inoculate stromal cells into the microcarriers 4) After the stromal cells proliferate to cover more than 80% of the microcarrier surface, the cells are treated to destroy mitosis and prevent cell proliferation; 5) Prepare a homogeneous hematopoietic stem cell culture medium; 6) Isolate PBMC from umbilical cord blood, sort out CD34+ cells after obtaining PBMC, and identify them by flow cytometry; 7) Add homogeneous hematopoietic stem cells to the sorted CD34+ cells and treated stromal cells in a three-dimensional culture system culture medium.

The hematopoietic stem cells are obtained by sorting from human umbilical cord blood, and the umbilical cord blood is obtained with the knowledge of the puerpera.

The three-dimensional cell culture materials include microcarriers, algae, nanometer materials and the like.

The stromal cells co-cultured with the cells include human umbilical cord mesenchymal stem cells, placental mesenchymal stem cells, AGM region cells, etc. or other related stromal cells that induce hematopoietic differentiation.

The method for disrupting cell mitosis and preventing cell proliferation includes mitomycin C treatment or radiation irradiation.

The culture medium refers to the small molecule-aryl hydrocarbon receptor antagonist StemRegenin-1 (SR1) added in IMDM supplemented with 10% FBS.

The culture medium refers to cytokines such as SCF, TPO, and Flt3L added to IMDM supplemented with 10% FBS.

Detection of CD34+ cell ratio by flow cytometry: Immediately after sorting and during expansion and culture, flow cytometry was used to detect the expression and proportion of CD34+ on the cell surface. The results show that after 10 days of culture in the present invention, the proliferation of CD34+ cells is nearly 20 times, and the positive rate of CD34+ cells is still very high (99%).

The present invention utilizes a three-dimensional culture system, combined with co-cultured stromal cells, under the joint action of SR1 and various cytokines, to promote the proliferation of umbilical cord blood hematopoietic stem cells, and establishes a rapid and stable method for proliferating umbilical cord blood hematopoietic stem cells. The present invention can obtain A large number of functional hematopoietic stem cells provide theoretical technology and a technical platform for clinically solving the bottleneck problem of insufficient cells in hematopoietic stem cell transplantation.

Description of drawings

In the following, umbilical cord mesenchymal stem cells are used as stromal cells, CULTISPHER-GL is used as a three-dimensional culture carrier, CD34+ cells are sorted by flow cytometry, and co-cultured with SR1 and cytokines is described.

Figure 1 is the growth state of umbilical cord mesenchymal stem cells extracted and cultured (P1 generation, 4×).

Figure 2 is the flow cytometry identification results of umbilical cord mesenchymal stem cells.

Figure 3 is microcarriers (10x) of treated uninoculated cells.

Fig. 4 is a microcarrier treated with mitomycin C after being inoculated with umbilical cord mesenchymal stem cells (placental blue staining, 10×).

Fig. 5 is a schematic diagram of flow cytometry sorting.

Figure 6 is a diagram of detection of CD34+ cells after sorting.

Figure 7 is the state of hematopoietic stem cells after 4 days of co-culture (10×).

Figure 8 shows the proportion of CD34+ cells detected by flow cytometry after 4 days of co-culture.

Figure 9 is the state of hematopoietic stem cells after 7 days of co-culture (10×).

Figure 10 shows the proportion of CD34+ cells detected by flow cytometry after 7 days of co-culture.

Figure 11 is the state of hematopoietic stem cells after co-culture for 10 days (10×).

Figure 12 shows the proportion of CD34+ cells detected by flow cytometry after 10 days of co-culture.

detailed description

The technical solutions in the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of the present invention.

A cell culture method for promoting rapid and stable proliferation of umbilical cord blood hematopoietic stem cells by using a three-dimensional cell culture system, comprising the following steps:

1. All materials, reagents, etc. in the examples can be obtained from commercial sources unless otherwise specified.

2. Extraction, culture, expansion and identification of stromal cells umbilical cord mesenchymal stem cells (the umbilical cords of newborns were obtained with the knowledge of the puerpera, and the puerpera was in good health.)

1) Take out the umbilical cord, soak it quickly in a small beaker pre-filled with 75% alcohol for 30 seconds to 1 minute, take it out and place it in a 150mm plate, wash it twice with PBS, and wash away the alcohol.

2) Cut the umbilical cord into 4-5cm pieces, wash with PBS 2-3 times, and remove residual blood in the blood vessel. Use toothed tweezers to tear each section of umbilical cord, carefully peel off the umbilical cord cortex and blood vessels (2 arteries and 1 vein, if the vein cannot be removed), then use tweezers to tear the umbilical cord into filaments, the thinner the better.

3) Cut the torn umbilical cord into pieces with ophthalmic scissors in a 100ml small beaker, about 1mm ³ or so, the more pieces the better.

4) Divide the shredded umbilical cord into two 50ml centrifuge tubes, add 15ml of 0.2% type I collagenase to each, and invert the two tubes repeatedly.

5) The 2 tubes of tissue after mixing were placed in a CO ₂ incubator at 37°C for overnight digestion, during which they were taken out and mixed several times by inversion.

6) After one digestion of type I collagenase (at this time, the 2 tubes of tissue are turbid, and some slightly larger tissue pieces are not completely digested, and the viscosity is very high), add 0.25% trypsin to the 2 tubes of tissue to make the final concentration of trypsin When it reaches 0.05%, put it into a CO ₂ incubator at 37°C to further digest the undigested tissue pieces.

7) Prepare 2 bottles of 0.05% trypsin (add 100ml 0.25% trypsin to 400ml PBS), 500ml each, and digest the tissues digested with 0.05% trypsin and 0.2% type I collagenase (at this time, the liquid is relatively clear and transparent, except Individual tissue blocks have been completely digested) directly added to the above 500ml liquid, 1 bottle per tube. Repeatedly invert and mix well, put into 37°C incubator, and digest for 60min.

8) Take out 2 bottles of liquid, blow gently until the tissue pieces are dispersed, and the liquid still has a certain viscosity at this time. Take a drop and observe the cell dispersion and cell volume under a microscope.

9) Transfer the liquid to a 50ml centrifuge tube, centrifuge at 3000rpm for 10min, discard the supernatant after centrifugation, and collect the precipitate into a new 50ml centrifuge tube.

10) Collect the digested cells in two 500ml bottles into four 50ml centrifuge tubes, add PBS to 45ml, pipette evenly, and centrifuge at 1500rpm for 6min.

11) Collect the centrifuged cells and resuspend them in the cell culture medium. Inoculate the resuspended cells into two 150mm culture dishes on average, add 20ml of culture medium to each dish, put them in a _CO2 incubator at 37°C, and keep them still for 3 days. Observe whether the culture medium becomes turbid (whether it is contaminated ) will do.

12) On the 3rd day after inoculation, take out the cells and observe the cell state under a microscope. The umbilical cord MSC cells grow adherently and are evenly distributed. Cells were then half changed.

13) When the cells grow to 90% of the culture dish, they are digested and passaged. Then the P2 generation cells were taken and identified by flow cytometry.

3. Three-dimensional culture cell scaffold processing

The microcarrier used in this example is the commercial product CULTISPHER-GL, and the treatment method is as follows: Weigh 0.1g of the microcarrier, add 20ml of PBS to soak for 3-4 hours, replace with a new 20ml of PBS and soak overnight, then 121°C, 0.1Mpa, high pressure Sterilize for 20 minutes. After sterilization, remove the PBS and add 20ml of complete medium to soak for later use.

4. Inoculation and treatment of three-dimensional co-culture matrix cells

Take umbilical cord mesenchymal stem cells in good growth state, wash them twice with PBS, add 2ml of trypsin to each bottle, digest for 2min, add 5ml of complete medium to stop the digestion, suck the digested cells into a centrifuge tube, and centrifuge at 1000 rpm for 5 minutes. After centrifugation, the cells were resuspended and counted by a cell counter. Take 4×10 ⁶ cells after counting and resuspend to 30ml. First mix the microcarriers and add them all into the three-dimensional culture bottle, then add the cells into the three-dimensional culture bottle, mix them well and put them in the incubator for culture, set the instrument at 35 rpm, run for 5 minutes, stop for 15 minutes, 12 cycles, Then add 50ml of culture medium to a final volume of 100ml, and set the instrument at 50 rpm for continuous operation. The final cell culture density was 4×10 ⁴ cells/ml. When the cells cover more than 80% of the surface of the microcarrier, add mitomycin C at a final concentration of 10 μg/ml to treat the cells for 2 hours, replace with a new medium, and continue culturing for later use.

5. Acquisition of Hematopoietic Stem Cells

In this example, the puerpera was in good health, and the umbilical cord blood was obtained with the knowledge of the puerpera.

1) Centrifuge the obtained cord blood at 3000 rpm for 10 minutes to remove serum.

2) Dilute the pellet after centrifugation to twice the volume before centrifugation with PBS.

3) Draw 30ml and slowly add to a 50ml centrifuge tube pre-filled with 15ml Ficoll.

4) After adding all the blood, centrifuge at 2000 rpm for 20 minutes, and slow down the speed at the end of the centrifugation.

5) Aspirate the second layer of buffy coat cell layer into a centrifuge tube, add 30ml PBS to wash twice.

6) After cell counting, 3 μl of CD34 antibody was added to every 10 ⁶ cells for staining, and the staining was performed at room temperature for 20 minutes. After staining, wash with PBS twice, adjust the concentration of resuspended cells to 10 ⁷ /ml, and sort. After sorting, the cell purity was detected by flow cytometry.

6. Inoculation and culture of hematopoietic stem cells

The sorted CD34+ cells were counted. Discard the medium in the three-dimensional culture flask, add 50ml of hematopoietic stem cell medium, take 1.5× ¹⁰⁶ CD34+ cells, resuspend them in 50ml of hematopoietic stem cell medium, add them to the three-dimensional culture flask for co-cultivation, and set the instrument at 50 rpm minutes, continuous operation. The umbilical cord blood hematopoietic stem cell medium is IMDM containing 10% FBS, and the final concentration is 1 μM small molecule-aromatic hydrocarbon receptor antagonist StemRegenin-1 (SR1), SCF100ng/ml, Flt3L 40ng/ml, TPO 2ng/ml, IL-6 20ng/ml. During the cell culture process, the cultured hematopoietic stem cells were half-changed on the 4th day, the 7th day, and the 10th day, and then after the cells were mixed, 2ml of the cell culture suspension was sucked out, and the microcarriers were screened out with a 200-mesh cell sieve. Into a 6-well plate to take pictures and count. Another 2ml was sucked out, 200-mesh cell sieve was used to remove microcarriers, the culture medium containing umbilical cord blood hematopoietic stem cells was centrifuged at 1000 rpm for 5 minutes, washed once with PBS, CD34 antibody was added, and stained at 4°C for 30 minutes. Wash once, add 200 μl of 2% paraformaldehyde to fix, and detect the percentage of CD34+ cells on a flow cytometer.

The results after 10 days of culture showed that the percentage of CD34+ cells and low expression was 99%, which was comparable to the percentage of CD34+ detected just after sorting; the cell concentration was 23.6×104 cells/ml, nearly ²⁰ times the cell concentration at the initial culture .

Claims (5)

1. A cell culture method utilizing a three-dimensional cell culture system to promote the rapid and stable proliferation of umbilical cord blood hematopoietic stem cells, characterized in that: realizing this invention mainly includes the following techniques and steps: Using the existing three-dimensional cell culture system, various cells related to hematopoiesis are used as stromal cells, and various three-dimensional materials that are harmless to cells are used as carriers, combined with various cytokines to proliferate and culture the sorted hematopoietic stem cells . After 7-10 days of proliferation and culture, a large number of hematopoietic stem cells with stable phenotypes were obtained. 2. a kind of cell culture method that utilizes three-dimensional cell culture system to promote the rapid and stable proliferation of umbilical cord blood hematopoietic stem cells according to claim 1, is characterized in that: The homogeneous hematopoietic stem cell culture medium used for co-culturing and proliferating hematopoietic stem cells was based on the addition of aryl hydrocarbon receptor antagonist StemRegenin-1 (SR1) at a final concentration of 1 μM in IMDM medium containing 10% FBS. 3. a kind of cell culture method that utilizes three-dimensional cell culture system to promote the rapid and stable proliferation of umbilical cord blood hematopoietic stem cells according to claim 1, is characterized in that: The cytokines added to the medium used in the proliferation and culture of umbilical cord blood hematopoietic stem cells are SCF, Flt3L, TPO, IL-6, and EPO. 4. a kind of cell culture method that utilizes three-dimensional cell culture system to promote the rapid and stable proliferation of umbilical cord blood hematopoietic stem cells according to claim 1, is characterized in that: The carriers used in the three-dimensional cell culture system are three-dimensional cell culture materials such as various seaweed materials, nanomaterials and macroporous gelatin materials. 5. a kind of cell culture method that utilizes three-dimensional cell culture system to promote the rapid and stable proliferation of umbilical cord blood hematopoietic stem cells according to claim 1, is characterized in that: The stromal cells used in the proliferation and culture of hematopoietic stem cells are human umbilical cord mesenchymal stem cells, placental mesenchymal stem cells, AGM cells or mouse bone marrow cells, AGM cells, mouse bone marrow cell lines or other cells related to hematopoiesis.