CN100384987C - A method for expanding hematopoietic stem cells under three-dimensional conditions - Google Patents

Abstract

The invention belongs to the fields of biotechnology and tissue engineering, in particular to a method for co-cultivating two kinds of cells under three-dimensional conditions. The method is characterized in that the trophoblasts are embedded with calcium alginate microgel beads, and then the embedded trophoblasts and hematopoietic stem cells are co-cultured in a rotating wall bioreactor to harvest the hematopoietic stem cells and the trophoblasts. The effects and benefits of the present invention are that the pores of the microgel beads can allow the important growth factors secreted by the trophoblasts to pass through the microgel beads to nourish the external hematopoietic stem cells, and at the same time the microgel beads effectively separate the cells of two different sources, not only It plays the role of immune isolation, and is also conducive to the separation and harvest of these two types of cells; the rotating wall bioreactor provides a three-dimensional suspension growth environment for hematopoietic stem cells, which effectively reduces the shear force on hematopoietic stem cells and is conducive to the growth of hematopoietic stem cells. Amplify.

Description

A method for expanding hematopoietic stem cells under three-dimensional conditions

technical field

The invention belongs to the fields of biotechnology and tissue engineering, in particular to a method for expanding hematopoietic stem cells under three-dimensional conditions.

Background technique

Cells are in a complex growth environment in the body, and there are important connections between different types of cells. Some cells need to rely on other cells to nourish and support them to varying degrees when cultured in vitro. The former are called trophoblast cells. The latter are called trophoblasts. This trophic effect is particularly important for the in vitro culture of stem cells, such as the in vitro culture of hematopoietic stem cells.

Hematopoietic stem cells have broad clinical application prospects. They can rebuild the hematopoietic system of patients after high-dose radiotherapy and chemotherapy, perform gene therapy and immunotherapy, and prepare mature blood products. However, in practical applications, there is a limitation on the number of hematopoietic stem cells, which requires large-scale expansion. Simulating the in vivo microenvironment has been considered as the best method to expand hematopoietic stem cells in vitro. Stromal cells are a kind of trophoblast cells that can nourish hematopoietic stem cells, and a variety of stromal cell lines have been used to support the expansion of hematopoietic stem cells in vitro. They can secrete a variety of known and unknown growth factors to maintain and expand hematopoietic stem cells, and avoid the gradual differentiation and apoptosis of hematopoietic stem cells during in vitro culture.

In terms of the means of co-cultivation of two kinds of cells, most studies have adopted the method of direct contact co-cultivation of stromal cells and hematopoietic stem cells, that is, first laying a layer of stromal cells on the bottom of the culture vessel, and then irradiating the stromal cells with a certain dose of radiation. Inhibit the activity of stromal cells, and then culture hematopoietic stem cells on the stromal cell layer. This direct contact culture method can support the nourishment of hematopoietic stem cells and promote the expansion of hematopoietic stem cells, but it will also make some hematopoietic stem cells adhere to the stromal cells, which is not conducive to the separation and harvest of the two cells; There is also the problem of immune rejection. At present, before the hematopoietic stem cells cultured in vitro are transfused into the human body, the stromal cells must be completely separated from the hematopoietic stem cells to ensure the biosafety of the implantation. Although some researchers use small-diameter omentum to separate the two types of cells in the co-culture system, which improves the problem of cell separation to a certain extent, such as Kawada et al. (Experimental Hematology, 27, 904, 1999) used A porous membrane separates the cord blood CD34 ⁺ cells from the murine stromal cells, but the villi of these two types of cells are still able to directly contact through the porous omentum, and the immune problem still exists. There is therefore a need to develop new methods for isolating heterologous cells in co-cultures.

In addition, most of the current co-culture of stromal cells and hematopoietic stem cells is carried out under static culture conditions, such as using well plates or culture flasks. Although static culture is convenient, it has the following disadvantages:

1) The lack of effective mixing of the culture medium leads to the formation of a concentration gradient in the culture medium of nutrients such as cytokines, dissolved oxygen, metabolites and other substances, as well as pH values;

2) Various parameters of the static culture system are generally difficult to monitor and control online;

3) Operations such as manual fluid change are required, increasing the risk of contamination;

4) The two-dimensional static culture is far away from the three-dimensional growth environment in the human body, which is easy to cause the differentiation of stem/progenitor cells to a certain extent.

Therefore, it is necessary to develop a suitable three-dimensional dynamic co-culture system to improve the culture environment to prepare hematopoietic stem cells with guaranteed quantity and quality. Perfusion (Palsson et al., Bio/Technology, 11, 368, 1993) and fixed-bed bioreactors (Meissner et al., Cytotechnology, 30, 227, 1999) that are often used for co-cultivation of stromal cells and hematopoietic stem cells, due to their internal The fluid shear force is relatively large, the degree of interaction between stromal cells and hematopoietic stem cells is limited, and the effect of expanding hematopoietic stem cells is not good.

Contents of the invention

The purpose of the present invention is to provide a method for expanding hematopoietic stem cells under three-dimensional conditions.

Technical scheme of the present invention comprises the following steps:

1. Provide trophoblasts. New Zealand white rabbits weighing about 2.5kg at four weeks old were killed by air injection, the femur and tibia were taken out, soaked in 75% alcohol for 30 minutes, and transferred to a sterile ultra-clean bench. Cut both ends of the bone with bone forceps, rinse the bone marrow cavity with PBS, and flush out the bone marrow. Add serum-free DMEM medium to dilute, and centrifuge at 1200rpm for 10 minutes. Discard the fat layer and add serum-free DMEM medium to dilute. Then it was mixed with Ficoll cell separation solution at a volume ratio of 1:1, and centrifuged at 3000 rpm for 30 minutes. Aspirate the interface layer, add serum-free DMEM medium to wash twice, and centrifuge at 1200rpm for 5 minutes. Remove the supernatant, add DMEM medium containing 20% fetal calf serum, adjust the cell concentration, and inoculate in a culture flask at a density of 5×10 ⁵ cells·cm ^-2 . After three days, the suspended cells were removed, and the medium was changed every five days in half. When the primary rabbit bone marrow mesenchymal stem cells reached 80% confluence at the bottom of the culture flask, they were digested with 0.05% trypsin (containing 0.02% EDTA) and subcultured at a ratio of 1:3.

2. Calcium alginate microgel beads embed trophoblasts. Prepare the fourth-generation rabbit bone marrow mesenchymal stem cell suspension with a density of 2.5×10 ⁵ cells mL ^-1 , mix it with 1.5% sodium alginate solution at a volume ratio of 1:4, and add it drop by drop through a 5 ^# needle Add to 1.5% calcium chloride solution, stir continuously, react for 10 minutes, filter with 20 mesh screen to obtain microgel beads, wash with PBS three times, and obtain calcium alginate with a diameter of 2 mm and embedding rabbit bone marrow mesenchymal stem cells Microgel beads. The preparation of small-diameter microgel beads requires the use of a high-voltage electrostatic generator. The preparation process is shown in Figure 2, wherein the diameter of the calcium alginate microgel beads is mainly determined by the diameter of the needle tip, voltage, distance between the two electrodes, and sodium alginate solution. Droplet flow rate to control.

3. Isolation of human umbilical cord blood mononuclear cells. Umbilical cord blood comes from healthy puerpera who gave birth at term or infants who delivered at term by caesarean section, and the average amount of raw blood collected each time was 50-100 mL. After the cord blood was collected, mononuclear cells were obtained by density gradient centrifugation with Ficoll cell separation medium with a density of 1.077 g·mL ^-1 . The volume ratio of Ficoll separation medium to cord blood was 1:1-1:2. The separated cells were centrifuged and washed twice with IMDM basal medium (1000 rpm, 5 min), and the cell density was adjusted for later use.

4. Prepare the rotating wall bioreactor. A rotating wall vessel (RWV) is mainly composed of two circulation loops: (1) medium circulation loop; (2) gas circulation loop. Specifically, the circulation of the culture medium is: the culture medium is sent to the oxygenator 10 by the peristaltic pump 11 for oxygenation, then enters the culture chamber 13 from the feed port at the left end of the RWV to provide nutrients for the cells, and the culture medium with reduced oxygen content flows from the right end of the RWV The discharge port enters the peristaltic pump 11, and then is sent into the oxygenator 10 for oxygenation, and the next round of circulation is started. The gas cycle is: the gas containing 5% CO ₂ and 95% air in the CO ₂ incubator 8 is sent by the air pump 9 to the oxygenator 10 to supplement oxygen for the culture medium, and then returns to the CO ₂ incubator 8 for continuous circulation. The RWV main part oxygenator 10 and the culture chamber 13 are placed in the heat preservation cover 15 outside the CO incubator ₈ . Before the experiment, use cleaning agent and soft brush to clean the various parts of RWV, and then rinse with ultrapure water three times. Each part was then soaked overnight in a 75% alcohol solution and rinsed three times with ultrapure water. Assemble the main part of the RWV heat preservation cover 15, wrap the main part, the connected external pipeline, and the oxygenator separately and autoclave (121°C, 30min), and dry for later use.

5. In a rotating wall bioreactor, trophoblast cells embedded in calcium alginate microgel beads and hematopoietic stem cells were co-cultured in three-dimensional suspension. Assemble the RWV culture system under aseptic conditions in the ultra-clean bench, fill with sterile PBS and circulate for one hour, release the PBS, inject the culture solution, turn on the temperature control switch in the console 12 to control the temperature at 37°C. Inoculate the human umbilical cord blood mononuclear cells with a density of 2-5×10 ⁵ cells·mL ^-1 and the trophoblast cell suspension embedded in calcium alginate microgel beads into the RWV culture chamber 13, cover the insulation cover 15, and turn on the Peristaltic pump 11 and air pump 9. After one hour, turn on the power switch of the adjustable speed motor 14 in the console 12, and make the inner and outer cylinders of the RWV cultivation chamber 13 rotate in the same direction and at the same angular velocity by adjusting the knob, and the whole system starts to work at this moment. The rotation speed of the culture chamber 13 is gradually increased, after 3rpm for one hour, 5rpm for 12 hours, and finally reaches and maintains at 6rpm, so that the cells and microgel beads are fully suspended.

6. Separate hematopoietic stem cells and microgel beads, and harvest hematopoietic stem cells. After the co-cultivation is over, the suspension in the reactor culture chamber 13 is drawn out with a syringe, and the suspension is added dropwise on the 20-mesh stainless steel screen after high temperature sterilization, and a sterile small beaker is placed under the screen. The microgel beads are trapped on the sieve, and the hematopoietic stem cell suspension passes through the sieve into the beaker. The hematopoietic stem cell suspension in the beaker was centrifuged at 1000 rpm for 5 minutes, the supernatant was discarded, 1 ml of culture solution was added, pipetting, counting, and inoculation into culture flasks for culture.

7. Dissolve microgel beads and harvest trophoblasts. The harvested microgel beads were rinsed three times with PBS, soaked in a sodium citrate solution with a concentration of 55mM, reacted for 10 minutes, centrifuged at 1500rpm for 10 minutes, discarded the supernatant, then added 1ml of culture medium, pipetted, and counted bone marrow mesenchymal cells. The number of mesenchymal stem cells was inoculated into culture flasks for culture.

Effect and advantage of the present invention are:

(1) The trophoblasts and hematopoietic stem cells can be co-cultured under three-dimensional conditions, so as to exert the nourishing and supporting effect of the trophoblasts on the hematopoietic stem cells.

(2) The two kinds of co-cultured cells can be isolated to avoid immune rejection between them, which is beneficial to the separation and harvest of the two kinds of cells after co-cultured.

(3) The rotating wall bioreactor significantly reduces the shear force of the cell growth environment, makes the cells grow under three-dimensional dynamic conditions all the time, and improves the cell culture effect.

(4) Using a method for amplifying hematopoietic stem cells under three-dimensional conditions of the present invention, dynamic co-cultivation of rabbit bone marrow mesenchymal stem cells embedded in calcium alginate microgel beads and human umbilical cord blood hematopoietic stem cells was carried out. The results showed that these two kinds of cells grew well in this culture system, and after seven days, the expansion folds of nucleated cells, CD34 ⁺ cells and myelomonocytic progenitor cells (colony-forming unit-granulocyte macrophage, CFU-GM) were respectively Up to 170 times, 24 times and 14 times.

Description of drawings

Fig. 1 is a flowchart of the operation of a method for expanding hematopoietic stem cells under three-dimensional conditions according to the present invention.

Fig. 2 is a schematic diagram of preparation of calcium alginate microgel beads by a method for expanding hematopoietic stem cells under three-dimensional conditions according to the present invention.

Fig. 3 is a process flow diagram of a rotary cell culture system that provides a three-dimensional suspension growth environment for cells and microgel beads in a method for expanding hematopoietic stem cells under three-dimensional conditions according to the present invention.

Fig. 4 is a schematic diagram of co-culture of trophoblasts and hematopoietic stem cells in a method for expanding hematopoietic stem cells under three-dimensional conditions according to the present invention.

Fig. 5 is a method for expanding hematopoietic stem cells under a three-dimensional condition of the present invention under static conditions, a microphotograph of calcium alginate microgel beads (2 mm in diameter) embedding mouse neural stem cells (A: Day 0 ; B: Day 9).

Fig. 6 is a micrograph of calcium alginate microgel beads (2 mm in diameter) embedding mouse calvarial osteoblasts under static conditions in a method for expanding hematopoietic stem cells under three-dimensional conditions according to the present invention.

Figure 7 is a method for expanding hematopoietic stem cells under a three-dimensional condition according to the present invention. Under static conditions, calcium alginate microgel beads (2 mm in diameter) embedding rabbit bone marrow mesenchymal stem cells and human umbilical cord blood hematopoietic stem cells Micrographs of co-cultivation for 168 hr (A: 100×; B: 200×).

In the figure: 1. Syringe; 2. Beaker; 3. Rotor; 4. Iron stand; 5. Anode; 6. Cathode; 7. High-voltage electrostatic generator; 8. CO ₂ incubator; 9. Air pump; 10. Oxygenation 11. Peristaltic pump; 12. Console; 13. Cultivation room; 14. Adjustable speed motor; 18. Calcium alginate microgel beads embedded with trophoblast cells and hematopoietic stem cells interacting with each other outside the microgel beads; 19. Hematopoietic stem cells outside the microgel beads; 20. Embedded in the microgel beads inner trophoblasts.

Detailed ways

Specific embodiments of the present invention will be described in detail below in conjunction with technical solutions and accompanying drawings.

Example 1:

This example is the in vitro culture and identification of mouse neural stem cells embedded in calcium alginate microgel beads.

In order to verify that cells can grow well in calcium alginate microgel beads, mouse neural stem cells were embedded in calcium alginate microgel beads with a diameter of 2 mm, and cultured and identified in vitro. Specifically, the sixth-generation mouse neural stem cell suspension was prepared, the total number of cells was 9×10 ⁵ , and the cell density was adjusted to 4×10 ⁵ cells·mL ^-1 . Mix this cell suspension with 1.5% sodium alginate solution at a volume ratio of 1:4 (the cell density at this time is 0.8×10 ⁵ cells·mL ^-1 ), and add dropwise to 1.5% CaCl through a 5 ^# needle ₂ solution, reacted for 10 minutes under stirring conditions, washed three times with PBS, and filtered through a sieve to obtain microgel beads. These microgel beads were equally divided into three parts and inoculated into culture flasks containing 3 ml of culture solution for culture. Take another mouse neural stem cell suspension with the same total number of cells, divide it into three parts evenly, and inoculate it in a culture bottle containing 3 ml of culture medium at a density of 1×10 ⁵ cells·mL ^-1 without embedding with microgel beads In , static culture was performed as a control group.

After culturing for 7 days, dissolve the calcium alginate microgel beads with 55mM sodium citrate solution, react for 10 minutes, centrifuge at 1500rpm for 10 minutes, discard the supernatant, add 1ml of medium, pipette, count, and immunohistochemical identification. At the same time, the neural stem cells in the control group were harvested, digested, pipetted, counted, and identified by immunohistochemistry.

The above experiments were repeated three times.

Experimental results: After 7 days of culture, the cell density of the control group reached 2×10 ⁵ cells·mL ^-1 , and the amplification factor was 2 times; the cell density of the calcium alginate microgel beads embedding group reached 1.58×10 ⁵ cells·mL ^-1 , the amplification factor was 1.98 times. Immunohistochemical identification revealed that the neural stem cells in the microgel beads, like the control neural stem cells, could express Nestin and differentiate into neurons and glial cells. Fig. 5 is a photomicrograph of calcium alginate microgel beads (2 mm in diameter) embedding mouse neural stem cells (A: Day 0; B: Day 9).

This example shows that cells can grow well in calcium alginate microgel beads; under static culture conditions, the growth of cells cultured in microgel beads is roughly the same as that of ordinary cultured cells, and at the same time, the mass transfer of calcium alginate microgel beads is verified The performance is good; since neural stem cells are very sensitive to shear force, it is suggested that under dynamic conditions, cells cultured in microgel beads may proliferate more.

Example 2:

This example is the capsule-breaking harvest rate when calcium alginate microgel beads embed mouse calvarial osteoblast cells.

Calcium alginate microgel beads with a diameter of 2 mm were used to embed mouse calvarial osteoblasts, and the cell harvest rate was determined by dissolving the beads with sodium citrate solution. Specifically, mix 1 ml of mouse calvarial osteoblasts with a density of 6×10 ⁵ cells·mL ^-1 with 4 ml of 1.5% sodium alginate solution, and add it dropwise into the 1.5% CaCl ₂ solution through a 5 ^# needle , reacted for 10 minutes under stirring conditions, and washed three times with PBS. Add 55 mM sodium citrate solution for 10 minutes, centrifuge at 1500 rpm for 10 minutes, discard the supernatant, add 1 ml of medium, pipette, count, and calculate the cell harvest rate.

The above experiments were repeated three times.

Experimental results: The cell harvest rates obtained in the three experiments were 86.7%, 79.2%, and 96.4%, respectively, and the average harvest rate reached 87.4%. This example illustrates that the method of harvesting cells by dissolving microgel beads with sodium citrate is feasible; after cell co-cultivation, most of the trophoblasts can be harvested by this method. Figure 6 is a photomicrograph of calcium alginate microgel beads (2 mm in diameter) embedding mouse calvarial osteoblasts.

Example 3:

This example is the cultivation of human umbilical cord blood hematopoietic stem cells in a rotating wall bioreactor.

In order to verify that the RWV culture system can cultivate stem cells that are more sensitive to the growth environment, in this example, human umbilical cord blood hematopoietic stem cells were cultured in the RWV culture system.

Isolation of mononuclear cells from human umbilical cord blood: umbilical cord blood comes from healthy mothers who gave birth at term or infants born at term by caesarean section, and the average amount of raw blood collected each time is 50-100mL. After the umbilical cord blood was collected, it was centrifuged with Ficoll lymphocyte separation medium with a density of 1.077g·mL ^-1 (2500r/min, 25min) to obtain mononuclear cells (containing about 1% CD34 ⁺ cells) and then cultured with IMDM The base was centrifuged and washed twice (1000rpmin, 5min) for later use.

Preparation of the bioreactor before the experiment: After cleaning each part of the RWV with a cleaning agent and a soft brush, rinse it three times with ultrapure water. Each part was soaked overnight in 75% alcohol solution and rinsed three times with ultrapure water. Assemble the main part of the RWV, wrap the main part, the connected external pipeline, and the oxygenator separately and autoclave (121°C, 30min), dry for later use.

Inoculation and cultivation of hematopoietic stem cells in the reactor: turn on the temperature control switch, control the temperature of the main part of the RWV in the heat preservation cover at 37°C, and inoculate the human umbilical cord blood mononuclear cells in the RWV at a density of 2×10 ⁵ cells·mL ^-1 In the cultivation room, turn on the peristaltic pump and the air pump. The culture medium is based on IMDM, supplemented with 10% fetal bovine serum, 10% horse serum and the following growth factors: SCF 16ng·mL ^-1 , FL 7.47ng·mL ^-1 , TPO 7.47ng·mL ^-1 , IL- 35.33ng·mL ^-1 , G-CSF 3.33ng·mL ^-1 , GM-CSF 2.13ng·mL ^-1 . The incubation chamber rotation speed of the reactor was maintained at 6 rpm after twelve hours via 0 rpm for one hour, 3 rpm for one hour, and 5 rpm for twelve hours.

After culturing for 7 days, samples were taken every day to count the number of nucleated cells, and CD34 ⁺ cell flow cytometry analysis and colony-forming unit-granulocyte/macrophage (CFU-GM) ability detection were performed at 0hr, 144hr and 197hr respectively. When it is detected that the number of nucleated cells in the culture system reaches or exceeds 1.5×10 ⁶ cells·mL ^-1 , extract part of the cell suspension and add fresh culture medium at the same time, so that the density of nucleated cells in the system does not exceed this value. Cells taken out were also considered to be still growing in the reactor and included in the expansion factor.

Culture results: after 7 days of three-dimensional suspension culture of human umbilical cord blood hematopoietic stem cells in the RWV culture system, nucleated cells expanded more than 400 times, CD34 ⁺ cells expanded more than 30 times, and CFU-GM progenitor cells expanded more than 20 times .

This example shows that: the rotating wall bioreactor can provide an excellent three-dimensional suspension growth environment for hematopoietic stem cells, and promote the expansion of hematopoietic stem cells; it is feasible to use this reactor to culture target cells in vitro.

Example 4:

This example is the in vitro expansion of human umbilical cord blood hematopoietic stem cells supported and nourished by rabbit bone marrow mesenchymal stem cells embedded in calcium alginate microgel beads under static conditions.

In order to verify that trophoblast cells embedded in calcium alginate microgel beads can support and nourish external hematopoietic stem cells, rabbit bone marrow mesenchymal stem cells embedded in calcium alginate microgel beads and human umbilical cord blood hematopoietic stem cells Co-cultivation was carried out under static conditions in six-well culture plates.

Isolation and culture of rabbit bone marrow mesenchymal stem cells: New Zealand white rabbits weighing about 2.5kg at four weeks old were killed by air injection, femur and tibia were taken out, soaked in 75% alcohol for 30 minutes, and transferred to a sterile ultra-clean bench Inside. Cut both ends of the bone with bone forceps, rinse the bone marrow cavity with PBS, and flush out the bone marrow. Add serum-free DMEM medium to dilute, and centrifuge at 1200rpm for 10 minutes. Discard the fat layer and add serum-free DMEM medium to dilute. Then it was mixed with Ficoll cell separation solution at a volume ratio of 1:1, and centrifuged at 3000 rpm for 30 minutes. Aspirate the interface layer, add serum-free DMEM medium to wash twice, and centrifuge at 1200rpm for 5 minutes. Remove the supernatant, add DMEM medium containing 20% fetal calf serum, adjust the cell concentration, and inoculate in a culture flask at a density of 5×10 ⁵ cells·cm ^-2 . After three days, the suspended cells were removed, and the medium was changed every five days in half. When the primary rabbit bone marrow mesenchymal stem cells reached 80% confluence at the bottom of the culture flask, they were digested with 0.05% trypsin (containing 0.02% EDTA) and subcultured at a ratio of 1:3.

Calcium alginate microgel beads embedding rabbit bone marrow mesenchymal stem cells: prepare the fourth-generation rabbit bone marrow mesenchymal stem cell suspension with a density of 2.5×10 ⁵ cells·mL ^-1 , mix with 1.5% sodium alginate solution at 1 : 4 volume ratio mixed evenly, dropwise added to 1.5% calcium chloride solution through a 5 ^# needle, stirred constantly, reacted for 10 minutes, filtered with a sieve to obtain microgel beads, washed three times with PBS, and obtained a diameter of 2 mm. Calcium alginate microgel beads embedded with rabbit bone marrow mesenchymal stem cells.

Separation of human umbilical cord blood mononuclear cells: same as in Example 3.

Co-cultivation of rabbit bone marrow mesenchymal stem cells and human umbilical cord blood mononuclear cells in a six-well culture plate: distribute microgel beads embedding rabbit bone marrow mesenchymal stem cells in a 6-well culture plate at a density of 3 Human umbilical cord blood mononuclear cells × 10 ⁵ cells·mL ^-1 were mixed and cultured in a 37°C, 5% CO ₂ incubator. The culture medium is based on IMDM, supplemented with 20% FBS, and supplemented with the following low-dose growth factors, SCF 16ng·mL ^-1 , FL 7.47ng·mL ^-1 , TPO 7.47ng·mL ^-1 , IL-3 5.33 ng·mL ^-1 , G-CSF 3.33 ng·mL ^-1 , GM-CSF 2.13 ng·mL ^-1 .

Initially, the ratio of the number of rabbit bone marrow mesenchymal stem cells embedded in microgel beads to the number of human umbilical cord blood mononuclear cells outside the microgel beads was about 4:100, that is, the ratio of mesenchymal stem cells to CD34 ⁺ cells was about 4:1 ( Initially, CD34 ⁺ cells accounted for about 1% of mononuclear cells), and 2 mL of culture medium was added to each well. 10% of the medium was changed every day, and the density of nucleated cells was counted, and CD34 ⁺ cell flow cytometry analysis and CFU-GM colony detection were carried out at 0hr, 72hr, 120hr and 168hr respectively.

Culture results: During the seven-day culture process, the expansion times of nucleated cells, CD34 ⁺ cells and CFU-GM in the co-culture system reached 9 times, 20 times and 9 times respectively. Fig. 7 is a photomicrograph (A: 100×; B: 200×) of the co-culture system cultured to 168 hours.

This example shows that: calcium alginate microgel beads are an effective means of co-cultivation of cells, can play a role in immune isolation, and are beneficial to the separation of mesenchymal stem cells and hematopoietic stem cells after co-culture; Rabbit bone marrow mesenchymal stem cells in beads can effectively support the expansion of human umbilical cord blood hematopoietic stem/progenitor cells under static conditions in vitro, indicating that the use of microgel beads to embed trophoblast cells does not hinder this trophobic effect; if combined with RWV The three-dimensional suspension growth environment of the culture system, microgel beads embedded trophoblasts will be able to better support and nourish the in vitro expansion of hematopoietic stem cells.

Example 5:

This example is the in vitro expansion of human umbilical cord blood hematopoietic stem cells supported and nourished by rabbit bone marrow mesenchymal stem cells embedded in calcium alginate microgel beads under dynamic conditions.

In order to verify that trophoblast cells embedded in calcium alginate microgel beads can support and nourish external hematopoietic stem cells, rabbit bone marrow mesenchymal stem cells embedded in calcium alginate microgel beads and human umbilical cord blood hematopoietic stem cells Co-cultivation was carried out under dynamic conditions in a rotating wall bioreactor.

Isolation and cultivation of rabbit bone marrow mesenchymal stem cells: same as in Example 4.

Calcium alginate microgel beads embed rabbit bone marrow mesenchymal stem cells: the same as in Example 4.

Separation of human umbilical cord blood mononuclear cells: same as in Example 3.

Preparation of the bioreactor before the experiment: same as Example 3.

Co-culture of rabbit bone marrow mesenchymal stem cells and human umbilical cord blood mononuclear cells in a rotating wall bioreactor: turn on the temperature control switch to control the temperature of the main part of the RWV in the heat preservation cover at 37°C, and embed 450 rabbit bone marrow mesenchymal stem cells Calcium alginate microgel beads and human umbilical cord blood mononuclear cells with a density of 3×10 ⁵ cells·mL ^-1 were inoculated in the RWV culture chamber, and the peristaltic pump and air pump were turned on. The culture medium is based on IMDM, and the following growth factors are added: SCF 16ng·mL ^-1 , FL 7.47ng·mL ^-1 , TPO 7.47ng·mL ^-1 , IL-35.33ng·mL ^-1 , G-CSF 3.33 ng·mL ^-1 , GM-CSF 2.13ng·mL ^-1 . The incubation chamber rotation speed of the reactor was maintained at 6 rpm after twelve hours via 0 rpm for one hour, 3 rpm for one hour, and 5 rpm for twelve hours.

After culturing for 7 days, samples were taken every day to count the number of nucleated cells, and CD34 ⁺ cell flow cytometry analysis and CFU-GM detection were performed at 0hr, 144hr and 197hr respectively. When it is detected that the number of nucleated cells in the culture system reaches or exceeds 1.5×10 ⁶ cells·mL ^-1 , extract part of the cell suspension and add fresh culture medium at the same time, so that the density of nucleated cells in the system does not exceed this value. Cells taken out were also considered to be still growing in the reactor and included in the expansion factor. After co-cultivation, hematopoietic cells and beads were isolated. The microgel beads embedding rabbit bone marrow mesenchymal stem cells were washed three times with PBS, soaked in 55mM sodium citrate solution, reacted for 10 minutes, harvested rabbit bone marrow mesenchymal stem cells, inoculated into culture flasks for culture.

Culture results: During the seven-day culture process, the expansion times of nucleated cells, CD34 ⁺ cells and CFU-GM in the co-culture system reached 170 times, 24 times and 14 times, respectively. Trophoblastic cells—the harvest rate of rabbit bone marrow mesenchymal stem cells reached about 90%.

This example illustrates that rabbit mesenchymal stem cells embedded in calcium alginate microgel beads in a rotating wall bioreactor effectively nourish and support the in vitro expansion of hematopoietic stem cells. Although the expansion factor of hematopoietic stem cells in this example is not as high as that in Example 3, the embedded trophoblasts are used instead of animal serum in this example, which is closer to clinical practice and has more medical value.

Although the present invention is described by taking rabbit bone marrow mesenchymal stem cells nourishing hematopoietic stem cells derived from human umbilical cord blood as an example, this description does not mean to limit the present invention. With reference to the description of the present invention, other types of trophoblasts, trophoblasts and other modifications of the embodiments are expected by those skilled in the art. Therefore, such modifications do not depart from the scope and spirit defined by the appended claims.

Claims (1)

1. A method for expanding hematopoietic stem cells under three-dimensional conditions, characterized in that the following steps: (1) Provide trophoblasts: trophoblasts are bone marrow mesenchymal stem cells from rabbit femur and tibia, seeded in DMEM medium containing 20% fetal bovine serum at a density of 5×10 ⁵ cells·cm ^-2 and cultivated. When the bottom of the culture bottle reaches 70% fusion, add 0.05% trypsin containing 0.02% EDTA to digest, and carry out conventional subculture according to the ratio of 1:3; (2) Calcium alginate microgel beads embedded trophoblasts: 1.5% sodium alginate solution and trophoblast suspension with a density of 2.5×10 ⁵ cells·mL ^-1 were mixed at a volume ratio of 4:1, and passed through 5# Add the needle dropwise into 1.5% calcium chloride solution, react for 10 minutes, and filter through a 20-mesh sieve to obtain calcium alginate microgel beads with a diameter of 2 mm and embedded with trophoblast cells; (3) Isolation of mononuclear cells from human umbilical cord blood: umbilical cord blood comes from healthy puerperas who gave birth at term and babies born at term by cesarean section. Based on 1000rpm, centrifuge and wash twice for 5min, adjust the cell density for later use; (4) Prepare the rotating wall bioreactor: the rotating wall bioreactor is mainly composed of two circulation loops: a medium circulation loop and a gas circulation loop; the medium circulation loop includes a peristaltic pump, an oxygenator, a culture chamber, a medium It is sent to the oxygenator by a peristaltic pump for oxygenation, and then enters the culture chamber from the feed port at the left end of the bioreactor to provide nutrients for the cells. The medium with reduced oxygen content enters the peristaltic pump from the feed port at the right end of the bioreactor, and then into Oxygen is filled in the oxygenator, and the next round of cycle begins; the gas circulation circuit includes a _CO2 incubator, an air pump, and an oxygenator. The gas containing 5% _CO2 and 95% air in the _CO2 incubator is sent into the oxygenator by the air pump. Oxygen supplements the culture medium in the oxygenator, and then returns to the _CO2 incubator for continuous circulation; the oxygenator and the culture chamber of the main part of the rotating wall bioreactor are placed in the insulation cover outside the _CO2 incubator, (5) In a rotating wall bioreactor, trophoblast cells and hematopoietic stem cells embedded in calcium alginate microgel beads are co-cultured in three-dimensional suspension: the inner and outer cylinders of the rotating wall bioreactor culture chamber rotate in the same direction and at the same angular speed, and the rotation speed is from Static increase gradually, after 0rpm for one hour, 3rpm for one hour and 5rpm for twelve hours, finally reach and maintain at 6rpm; the seeding density of human cord blood mononuclear cells in the rotating wall bioreactor culture chamber is 2～5× 10 ⁵ cells·mL ^-1 ; (6) Separating hematopoietic stem cells and microgel beads, and harvesting hematopoietic stem cells: use a 20-mesh stainless steel screen to intercept the calcium alginate microgel beads embedded in bone marrow mesenchymal stem cells, collect the filtrate—hematopoietic stem cell suspension, and centrifuge at 1000rpm 5 minutes, harvest hematopoietic stem cells; (7) Dissolving microgel beads and harvesting trophoblasts: the calcium alginate microgel beads embedded with trophoblast cells were dissolved in 55 mM sodium citrate solution for 10 minutes, centrifuged at 1500 rpm for 10 minutes, and trophoblasts were harvested.

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