CN111334431A

CN111334431A - Mesenchymal stem cell culture bioreactor and use method thereof

Info

Publication number: CN111334431A
Application number: CN202010137724.6A
Authority: CN
Inventors: 韩荣成; 杜如龙
Original assignee: Hanlang Beijing Technology Co ltd
Current assignee: Hanlang Beijing Technology Co ltd
Priority date: 2020-03-02
Filing date: 2020-03-02
Publication date: 2020-06-26

Abstract

The invention relates to a cell culture device, in particular to a mesenchymal stem cell culture bioreactor and a use method thereof. The mesenchymal stem cell culture bioreactor comprises a shell, wherein a culture medium cavity and a cell suspension cavity are arranged in the shell; the culture medium chamber is composed of a plurality of internal pipelines of hollow fibers with gaps; the cell suspension chamber is composed of a gap between the hollow fibers and the inner wall of the shell, and a plurality of microspheres are arranged in the cell suspension chamber. Compared with the existing mesenchymal stem cell culture mode, the invention simulates the environment of in vivo cell growth by utilizing the mesenchymal stem cell culture bioreactor, and realizes the large-scale in vitro cell three-dimensional culture.

Description

Mesenchymal stem cell culture bioreactor and use method thereof

Technical Field

The invention relates to a cell culture device, in particular to a mesenchymal stem cell culture bioreactor and a use method thereof.

Background

Cell culture technology lays the foundation for studying the structure, function, differentiation of animal cells and producing many important biological products such as proteins, cytokines, vaccines, hormones, antibodies, interferons, nucleic acids, etc. The clinical mesenchymal stem cell treatment and the preparation of the mesenchymal stem cell biological products can not be separated from large-scale cell culture.

At present, the conventional cell culture methods are mainly of the following types: monolayer adherent culture, hollow fiber three-dimensional culture and microsphere suspension culture. The monolayer adherent culture is planar 2D culture, is usually adherent culture in a culture dish, a culture plate, a culture square bottle and a roller bottle and has the defects of low culture density, large batch difference, high manpower and cost, large occupied area and the like. The hollow fiber culture and the microsphere suspension culture are more suitable for large-scale three-dimensional culture. In the microsphere culture, microparticles are used as carriers to which cells are attached, and the microparticles are suspended in a culture solution under agitation to allow the cells to grow as a monolayer on the surface of the carriers. Traditional microsphere suspension culture has better cell adherence effect, but adherence cells face the problems of large shearing force, low dissolved oxygen, poor amplification effect and the like. The hollow fiber cell culture refers to culture of cells by using hollow fiber ultrafiltration or microfiltration membrane center or outside, imitating blood vessels. The traditional three-dimensional culture of the hollow fibers also has a plurality of defects, such as the structure that the hollow fibers are longitudinally bundled and densely arranged, so that a good cell growth environment cannot be provided, and the three-dimensional culture of cells is not facilitated; cells are unevenly distributed, are easy to aggregate and grow at certain positions, and can not effectively utilize culture space; when the cells grow adherently, the hollow fiber membrane pores are easy to block, and the exchange of nutrient substances and cell metabolic waste is influenced. The defects of the conventional mesenchymal stem cell culture mode lead to the problems of low cell yield, poor activity, cell function loss, high cost, poor batch stability and the like, thereby obviously influencing cell acquisition and extraction of biological products.

Therefore, how to improve the defects of the traditional mesenchymal stem cell culture mode becomes one of the key factors for realizing high-quality and large-scale cell culture.

Disclosure of Invention

Technical problem to be solved

In order to overcome the defects of the existing mesenchymal stem cell culture mode, the invention provides a three-dimensional cell culture bioreactor with simple structure and convenient use, namely a mesenchymal stem cell culture bioreactor and a use method thereof.

(II) technical scheme

The invention firstly provides a mesenchymal stem cell culture bioreactor which comprises a shell, wherein a culture medium cavity and a cell suspension cavity are arranged in the shell;

the culture medium chamber is composed of a plurality of internal pipelines of hollow fibers with gaps, and gaps are arranged among the hollow fibers; the membrane aperture of the hollow fiber material is 0.05-5 μm;

the cell suspension chamber is composed of a gap between the hollow fibers and the inner wall of the shell, and a plurality of microspheres are arranged in the cell suspension chamber;

an opening I for the culture medium to come in and go out and an opening II for the cell suspension to come in and go out are respectively arranged on the surface of the shell, the culture medium chamber is communicated with the opening I, and the cell suspension chamber is communicated with the opening II.

The invention discovers that the three-dimensional growth space of the cells can be formed on the outer membrane surface of the hollow fiber, the surface of the microsphere and the inner wall of the reactor shell together through the arrangement, and a good nutrition exchange channel is provided for the cells through the hollow fiber with the specific membrane aperture, so that the growth of the mesenchymal stem cells is facilitated.

In order to research the space environment and the nutrient exchange rate which are more beneficial to the growth of the mesenchymal stem cells, the invention researches and tests related factors and discovers the following more key factors and the preferable scheme thereof:

preferably, the hollow fiber is made of a material having a membrane pore diameter of 0.22 to 1 μm.

Preferably, the outer diameter of the hollow fiber is 50-500 μm, and the thickness of the pipe wall is 6-14% of the outer diameter; the preferred outer diameter is 100-300 μm, and the thickness of the pipe wall is 8-12% of the outer diameter.

Preferably, the gap between the hollow fibers and the inner wall of the housing are 100 to 1000 μm, and the ratio of the gap to the outer diameter of the hollow fibers is 2 to 3: 1.

Preferably, the microspheres have a diameter of 50 to 500 μm, and the ratio of the diameter of the microspheres to the gaps is 1:4 to 6.

Preferably, when the hollow fibers are made of hydrophobic materials, the blockage of fiber membrane pores caused by the adherent growth of cells can be reduced, and the exchange rate of nutrient substances, gas and metabolic waste is improved. In a great number of attempts, polyvinylidene fluoride is found to be more beneficial for the culture of mesenchymal stem cells.

Preferably, the microsphere is a dextran hydrogel microsphere, and further preferably, when the outer surface of the microsphere is coated with a vitronectin or polypeptide layer, the surface characteristics of the microsphere can be changed, so that cells can be attached to the microsphere more easily.

Preferably, the material of the shell is organic glass or stainless steel.

The opening I can be an opening for the culture medium to enter and exit. To avoid cross-contamination, it is preferred that the housing comprises a medium inlet and a medium outlet, which are separately provided at different positions on the surface of the housing. The culture medium inlet and the culture medium outlet may be provided only one, or a plurality of culture medium inlets and culture medium outlets may be provided, respectively, without further limitation.

Similarly, the opening II can be an opening for the entrance and exit of the cell suspension. Preferably, when the opening II comprises a cell suspension inlet and a cell suspension outlet, which are separately disposed at different positions on the surface of the housing, not only can cross contamination be reduced, but also a detection device can be disposed at the cell suspension outlet to facilitate sampling and monitoring of the cell state based on the cell suspension. The cell suspension inlet and the cell suspension outlet may be provided only one, or a plurality of cell suspension inlets and cell suspension outlets may be provided, respectively, and are not further limited herein.

Preferably, the relative distance of the cell suspension inlet to the medium inlet is less than the relative distance of the cell suspension inlet to the medium outlet; the relative distance between the cell suspension outlet and the culture medium outlet is smaller than the relative distance between the cell suspension outlet and the culture medium inlet.

The hollow fiber of the present invention may have various shapes as long as it has a gap and is disposed to communicate with the opening I. Preferably, the housing is cylindrical, and the hollow fibers are arranged in parallel with the axis of the housing.

In order to increase the lifespan of the mesenchymal stem cell culture bioreactor, it is preferable that the microspheres are accessible through the opening II to facilitate sterilization and coating processes, so that they can be recycled.

The invention further provides a use method of the mesenchymal stem cell culture bioreactor, which comprises the following steps:

(1) injecting the cell suspension into the cell suspension chamber from the opening II, and placing the cell suspension in a cell culture box for standing so as to make the cells adhere to the wall;

(2) injecting a culture medium into the culture medium chamber from the opening I, and periodically replacing the culture medium;

(3) after a period of incubation, collecting cells and/or cell products from the cell suspension chamber through the opening II;

when the microspheres can enter and exit through the opening II, before the step (1), the method further comprises: injecting said microspheres into said cell suspension chamber through said opening II; more preferably, the microspheres after sterilization are coated with vitronectin or polypeptide prior to injection into the cell suspension chamber.

(III) advantageous effects

Compared with the existing mesenchymal stem cell culture mode, the invention simulates the environment of in vivo cell growth by utilizing the mesenchymal stem cell culture bioreactor, and realizes the three-dimensional culture of large-scale in vitro cells; the material exchange efficiency is high, which is beneficial to the rapid exchange of nutrient substances and cell metabolic waste; the shearing force borne by the cells is small, the cell morphology is more complete, and the cell activity is higher; meanwhile, a serum-free culture medium can be adopted in the mesenchymal stem cell culture process, so that the risk of animal-derived mycoplasma and virus infection is avoided.

Drawings

Fig. 1 is a schematic structural view of a longitudinal cross-sectional view of a mesenchymal stem cell culture bioreactor in example 1 of the present invention;

fig. 2 is a schematic structure of a transverse sectional view of a mesenchymal stem cell culture bioreactor in example 1 of the present invention;

FIG. 3 is a statistic result of the yields of umbilical cord mesenchymal stem cells of examples and comparative examples;

FIG. 4 shows the expression and purity of the surface markers of umbilical cord mesenchymal stem cells of example 1, wherein the filled cells represent CD34, CD45, CD73, CD90 and CD105, and the non-filled cells represent isotype control;

FIG. 5 shows the expression and purity of the surface markers of umbilical cord mesenchymal stem cells in comparative example 1, wherein the filled cells represent CD34, CD45, CD73, CD90 and CD105, and the unfilled cells represent isotype control;

FIG. 6 shows the expression and purity of the surface markers of umbilical cord mesenchymal stem cells in comparative example 2, wherein the filled cells represent CD34, CD45, CD73, CD90 and CD105, and the unfilled cells represent isotype control;

FIG. 7 shows microscopic results of cultured cells obtained in example 1;

FIG. 8 is a microscopic examination result of the cultured cells obtained in comparative example 1;

FIG. 9 shows microscopic results of cultured cells obtained in comparative example 2.

In FIGS. 1 to 2: 1. a housing; 2. an end cap; 3. a hollow fiber; 4. microspheres; 5. a cell suspension inlet; 6. a cell suspension outlet; 7. a culture medium inlet; 8. and (5) a culture medium outlet.

Detailed Description

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.

The examples do not show the specific techniques or conditions, according to the technical or conditions described in the literature in the field, or according to the product specifications. The reagents or instruments used are conventional products available from regular distributors, not indicated by the manufacturer.

Example 1

The embodiment provides a mesenchymal stem cell culture bioreactor, which is shown in a longitudinal sectional view structural schematic diagram in figure 1 and is shown in a transverse sectional view structural schematic diagram in figure 2, and comprises a cylindrical transparent organic glass reactor shell 1 and two end covers 2. The side wall of the reactor shell 1 is oppositely provided with a cell suspension inlet 5 and a cell suspension outlet 6, the end cover 2 close to one side of the cell suspension inlet 5 is provided with a culture medium inlet 7, and the end cover 2 at the other side is provided with a culture medium outlet 8.

The reactor shell 1 is a cylindrical transparent organic glass reactor shell with the length of 30cm, the inner diameter of 5cm and the wall thickness of 0.3cm, a plurality of longitudinal hollow fibers 3 (parallel to the axis of the shell 1) arranged in the reactor shell are polyvinylidene fluoride hydrophobic hollow fibers with the aperture of 0.45 mu m, the outer diameter of 200 mu m and the pipe wall thickness of 20 mu m, and the reactor shell has good semi-permeability. The hollow fiber 3 is internally provided with a cell culture medium channel, one end of the hollow fiber is connected with a cell culture medium inlet 7, the other end of the hollow fiber is connected with a culture medium outlet 8, and the cell culture medium channel jointly forms a culture medium chamber.

The gap between the hollow fibers 3 is 500 μm, the gap between the hollow fibers 3 and the inner wall of the shell 1 is 500 μm, and the gap between the hollow fibers 3 and the inner wall of the reactor shell 1 jointly form a cell suspension chamber, i.e., a three-dimensional cell growth space. The cell suspension chamber is filled with microspheres 4, the microspheres 4 are dextran hydrogel microspheres with the diameter of 100 mu m, and the outer surfaces of the microspheres 4 are coated with human recombinant vitronectin (rhVTN). The cell suspension chamber is in communication with a cell suspension inlet 5 and a cell suspension outlet 6.

The mesenchymal stem cell culture bioreactor in the embodiment is used for umbilical cord mesenchymal stem cell culture, and the specific use process is as follows:

1. preparation of the reactor

(1) 10g of microspheres 4 were soaked in PBS overnight and then autoclaved.

(2) After autoclaving microspheres 4 were washed with sterile PBS, human recombinant vitronectin (rhVTN, recombinant human vitronectin) was diluted to a concentration of 100 μ g/mL with 10mL of medium and the microspheres were coated overnight in a 37 ℃ cell incubator to change the surface characteristics and make the cells easier to adhere to the wall.

(3) The coated microspheres 4 are injected into the cell suspension chamber through the cell suspension inlet 5, and turned upside down several times, so that the microspheres are uniformly distributed in the cell suspension chamber.

2. Inoculating cells, selecting umbilical cord mesenchymal stem cells as the mesenchymal stem cells,

(1) suspending umbilical cord mesenchymal stem cells to 1 × 10 using 10mL of medium⁶cells/mL。

(2) Injecting the umbilical cord mesenchymal stem cell suspension into the cell suspension chamber from a cell suspension inlet 5, then supplementing about 40mL of culture medium until the cell suspension overflows from a cell suspension outlet 6, then repeatedly reversing for several times to fully mix the cells, and placing the cells in a cell culture box for standing for 60 minutes to allow the cells to adhere to the wall.

3. Fresh medium is pumped into the medium chamber from medium inlet 7 using a peristaltic pump, the medium flows through the inner tube of hollow fibre 3 to medium outlet 8 out of the reactor, exchange of nutrients and cellular metabolic waste is achieved through the hollow fibre membrane.

4. After 14 days of culturing, the cells and cell products were collected from the reactor.

Example 2

This example provides a mesenchymal stem cell culture bioreactor, which is different from example 1 in that: the gap between the hollow fibers 3 was 150 μm, and at this time, the ratio of the gap to the outer diameter of the hollow fibers 3 was 3: 4.

The mesenchymal stem cell culture bioreactor is used for culturing umbilical cord mesenchymal stem cells by adopting the same method as the example 1, and collecting cells and cell products.

Example 3

This example provides a mesenchymal stem cell culture bioreactor, which is different from example 1 in that: the gap between the hollow fibers 3 was 1000 μm, and at this time, the ratio of the gap to the outer diameter of the hollow fibers 3 was 5: 1.

Example 4

This example provides a mesenchymal stem cell culture bioreactor, which is different from example 1 in that: the microspheres 4 have a diameter of 50 μm, at which point the ratio of the microsphere diameter to the gap is 1: 10.

Example 5

This example provides a mesenchymal stem cell culture bioreactor, which is different from example 1 in that: the microspheres 4 had a diameter of 250 μm, at which point the ratio of the microsphere diameter to the gap was 1: 2.

Comparative example 1

Culturing umbilical cord mesenchymal stem cells with the same number as that in example 1 by adopting a traditional hollow fiber three-dimensional culture mode without microspheres, and collecting the cells and cell products from a reactor after culturing for the same time as that in example 1.

Comparative example 2

The conventional microsphere suspension culture mode is adopted, microspheres with the same weight of 10g are taken for suspension culture, umbilical cord mesenchymal stem cells with the same number as that of the umbilical cord mesenchymal stem cells in the example 1 are cultured, and after the culture for the same time as that of the example 1, the cells and cell products are collected from a reactor.

Comparative example 3

This comparative example provides a mesenchymal stem cell culture bioreactor, which is different from example 1 in that: the material of the hollow fiber 3 has a membrane pore diameter of 0.01 μm.

Comparative example 4

This comparative example provides a mesenchymal stem cell culture bioreactor, which is different from example 1 in that: the membrane pore diameter of the material of the hollow fiber 3 is 20 μm.

Test example 1 Total amount of cells

3 independent repeated experiments are carried out on the examples and the comparative examples, total cells cultured after 14 days of cell culture are respectively obtained, the total amount of the cells of each example or the comparative example is obtained through cell counting calculation, the average value and the variance of the total amount of the cells of each example or the comparative example in the 3 independent repeated experiments are taken to be used for making a bar chart, and the result is detailed as shown in figure 3. In fig. 3, N is 3, the value represents the average ± SD of the total number of cells collected in each example or comparative example, and P is <0.01, all of which are compared with example 1.

As can be seen from FIG. 3, example 1, in which the same number of umbilical cord mesenchymal stem cells were inoculated and cultured for 14 days, can collect about 1.31 × 10⁹The number of cells obtained from the examples (examples 2 to 5) in which the hollow fiber space or the microsphere exceeds the preferred range of the present invention is reduced to various degrees; the number of cells obtained by the traditional hollow fiber three-dimensional culture (comparative example 1) and microsphere suspension culture (comparative example 2) is obviously reduced; in addition, when the pore diameter of the hollow fiber membrane is beyond the range of the invention (comparative examples 3-4), the number of the obtained cells is also obviously reduced. Therefore, the three-dimensional culture mode of the hollow fiber combined microspheres, which is defined by the invention, greatly improves the yield of the umbilical cord mesenchymal stem cells.

Experimental example 2 cell purity and mesenchymal surface marker expression

In 2006, the international association for cell therapy stipulates that mesenchymal stem cells need to express cell surface markers CD105, CD90 and CD73, and the positive rate is more than or equal to 95%; and does not express cell surface markers CD45 and CD34, and the positive rate is less than 2%. And analyzing the expression condition of the mesenchymal stem cell surface marker and the purity and the homogeneity of the cell by a flow cytometer.

Cells were cultured in examples and comparative examples, pancreatin was added, the cells were allowed to stand at 37 ℃ for 2 to 3min (observation of cell state), when the cells became round, digestion was terminated by adding an equal volume of complete culture medium, centrifugation was carried out at 1200rpm for 3min, and the supernatant was discarded.

Cells were washed by adding 2mL of flow-staining buffer (eBioscience, 00-4222-26), centrifuged at 1200rpm for 3min, the supernatant was discarded, and repeated 3 times.

Cell division 1 × 10⁶Tube, 5 μ L of antibody or isotype control antibody (Mouse anti-Hu-CD105SN6 PE, eBioscience, 12-1057-42; Mouse anti-Hu-CD 905E 10 FITC, eBioscience, 11-0909-42; Mouse anti-Hu-CD73 AD2 FITC, eBioscience, 11-0739-42; Mouse anti-Hu-CD45HI30 FITC, eBioscience, 11-0459-42; Mouse anti-Hu-CD 344H 11 FITC, eBioscience, 11-0349-42; Mouse IgG1Kappa isopype Ct FITC, eBioscience, 11-4714-82; Mouse IgG1 kappat, Ctpype PE 12-4782), incubated at room temperature, protected from light.

Cells were washed by adding 2mL of flow-through staining buffer per tube, centrifuging at 1200rpm for 3min, discarding the supernatant, repeating 3 times, and washing away unbound antibody.

Adding 0.5mL of flow type staining buffer solution into each tube to resuspend the cells, placing the cells into a flow type detection tube, detecting by using a flow cytometer, and analyzing the detection result by using related software, wherein the result is shown in the attached figure 4-6 in detail.

Flow results show that the umbilical cord mesenchymal stem cells cultured in example 1 express positive mesenchymal stem cell surface markers CD73, CD90 and CD105, do not express mesenchymal stem cell surface markers CD34 and CD45 at all, and have extremely high cell purity and uniformity; the expressions of the positive markers of the umbilical cord mesenchymal stem cells cultured in the traditional hollow fiber three-dimensional mode (comparative example 1) and the microsphere suspension mode (comparative example 2) are attenuated to different degrees, the proportion of the positive cells is obviously reduced, and the cell purity and uniformity are obviously reduced, which shows that the dryness of the umbilical cord mesenchymal stem cells cultured by the traditional method is attenuated to different degrees, and the clinical application and scientific research of the obtained umbilical cord mesenchymal stem cells are seriously influenced.

Test example 3 morphological characteristics of cells

In order to further observe morphological characteristics of the cultured cells obtained in each example and comparative example, equal volume amounts of the cultured cells of the examples and comparative examples were respectively taken and placed in a culture dish for adherent culture. After 2 days of adherent culture, the growth state and morphological features of the cells of each example and comparative example were observed under a microscope.

And (3) microscopic examination result display: the umbilical cord mesenchymal stem cells obtained in example 1 (figure 7) and comparative example 1 (figure 8) are in a typical fibroblast long spindle shape, cells grow in a vortex or radial colony shape, no obvious particles are on the cell surface, and the suspension debris of dead cells is very little; however, comparative example 1 obtained a smaller number of cells and the proliferation potency of the cells was weaker than example 1; the cells obtained in comparative example 2 (fig. 9) are flat and grow in an aggregated manner, a large number of particles are on the cell surface, the number of dead cell suspension fragments is large, and the cell state is obviously poor.

Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Claims

1. A mesenchymal stem cell culture bioreactor is characterized by comprising a shell, wherein a culture medium chamber and a cell suspension chamber are arranged in the shell;

the culture medium chamber is composed of a plurality of hollow fiber internal pipelines, and gaps are arranged among the hollow fibers; the membrane aperture of the hollow fiber material is 0.05-5 μm;

2. The mesenchymal stem cell culture bioreactor according to claim 1, wherein the hollow fiber is made of a material having a membrane pore size of 0.22 to 1 μm.

3. The mesenchymal stem cell culture bioreactor of claim 1 or 2, wherein the hollow fiber has an outer diameter of 50 to 500 μ ι η and a tube wall thickness of 6 to 14% of the outer diameter; the preferred outer diameter is 100-300 μm, and the thickness of the pipe wall is 8-12% of the outer diameter.

4. Mesenchymal stem cell culture bioreactor according to any of claims 1 to 3, wherein the gaps between the hollow fibres and the inner wall of the shell are between 100 μm and 1000 μm, preferably the ratio of the gap to the outer diameter of the hollow fibres is between 2 and 3: 1.

5. Mesenchymal stem cell culture bioreactor according to any of claims 1 to 4, wherein the microspheres have a diameter of from 50 to 500 μm, preferably the ratio of the diameter of the microspheres to the interstices is from 1:4 to 6.

6. The mesenchymal stem cell culture bioreactor according to any one of claims 1 to 5, wherein the hollow fiber is made of a hydrophobic material, preferably polyvinylidene fluoride;

and/or, the microsphere is a dextran hydrogel microsphere, preferably the microsphere is coated with a vitronectin layer or a polypeptide layer on the outer surface;

and/or the shell is made of organic glass or stainless steel.

7. The mesenchymal stem cell culture bioreactor of any claim 1-6, wherein the opening I comprises a culture medium inlet and a culture medium outlet, the culture medium inlet and the culture medium outlet are respectively provided at different positions on the surface of the shell;

and/or the opening II comprises a cell suspension inlet and a cell suspension outlet which are respectively arranged at different positions on the surface of the shell.

8. Mesenchymal stem cell culture bioreactor of any claim 1 to 7, wherein the shell is cylindrical and the hollow fibres are arranged parallel to the axis of the shell.

9. Mesenchymal stem cell culture bioreactor according to any of claims 1 to 8, wherein the microspheres are accessible through the opening II.

10. The method of using a mesenchymal stem cell culture bioreactor of any of claims 1-9, comprising:

preferably, when the microspheres can enter and exit through the opening II, the method further comprises, before the step (1): injecting said microspheres into said cell suspension chamber through said opening II; more preferably, the microspheres after sterilization are coated with vitronectin or polypeptide prior to injection into the cell suspension chamber.