CN104894063A - Stem cell large-scale culture method - Google Patents

Abstract

A method for large-scale cultivation of stem cells. PNIPAAm is used in the cultivation process. The macromolecular chain of poly-N isopropylacrylamide (PNIPAAm) has both a hydrophilic amido group and a hydrophobic isopropyl group. The aqueous solution has the lowest The critical temperature (LCST) is 32°C, it can be cross-linked into a hydrogel at 37°C, and it is a linear aqueous solution at room temperature. Using the property of PNIPAAm to undergo a reversible phase transition near the LCST, PNIPAAm can be designed as a molecular switch, combined with stem cell culture, and the temperature sensitivity of poly-N-isopropylacrylamide (PNIPAAm) can be used to make cells stick when they are above the critical temperature. It grows on the surface of the material, and the cells detach when the temperature is below the critical temperature, which changes the traditional method of enzymatic detachment of cells, and the cells are in the three-dimensional space of the hydrogel, and the cell growth state is good and the state culture efficiency is increased.

Description

A method for large-scale culture of stem cells

technical field

The invention relates to the field of cell culture in biotechnology, in particular to a method for large-scale culture of stem cells.

Background technique

Stem cells are a type of pluripotent cells with self-replicating ability. Under certain conditions, it can differentiate into a variety of functional cells. Stem cells are divided into embryonic stem cells and adult stem cells according to their developmental stage. Stem cells are divided into three categories according to their developmental potential: totipotent stem cells, pluripotent stem cells and unipotent stem cells. Stem cells are not fully differentiated and immature cells that have the potential to regenerate various tissues and organs and the human body. called "universal cells".

In order to make full use of the various potentials of stem cells, the cultivation of stem cells has become a key point. There are many ways of traditional stem cell cultivation. Finally, there are many disadvantages. A very big problem is that the traditional culture method is aimed at the characteristics of adherent growth of stem cells during culture. Therefore, during culture, such as drum culture, stem cells can only adhere to the wall of the drum for a while. layer, and this layer should not be too thick, which greatly restricts the cultivation of stem cells on a larger scale, and also limits the large-scale output of stem cells. Therefore, in order to increase the large-scale culture of stem cells, it is necessary to improve the method of culture.

Hydrogel 3D cell culture is based on traditional 2D cell culture, adding a dimension in the form of hydrogel (hydrogel) or solid scaffold, so that cells grow in three-dimensional space like in vivo. The advantages of 3D over 2D cell culture are obvious: it is closer to the in vivo state of cells; it can increase the production of cytokines, antibodies and other biomolecules, and cells grow healthier in a 3D environment than in a 2D environment; in 3D cell culture In the system, stem cells can be differentiated in an orderly manner; experimental data are more reliable, such as gene expression profiles, cell activities, etc., are more consistent with in vivo research data; it can reduce the cost and cycle of new drug development; it can replace part of animal experiments. Moreover, it can improve the efficiency of cell culture and make large-scale culture of stem cells a reality.

For this reason, nanofiber scaffolds and porous scaffolds have appeared on the market for 3D culture of stem cells. However, due to the materials of the nanofiber scaffolds and porous scaffolds, on the one hand, the cost is high; Capacitance and so on are still lacking.

Contents of the invention

The invention proposes a method for large-scale cultivation of stem cells, which can realize large-scale cultivation of stem cells, is suitable for industrial application of stem cell cultivation, and has great popularization value.

A method for large-scale cultivation of stem cells, characterized in that PNIPAAm (poly-N isopropylacrylamide) is used in the cultivation process.

Further, the PNIPAAm serves as a carrier matrix for stem cell growth during the culture process.

Further, during the cultivation process, different temperature gradients are set, and the PNIPAAm is in different phase states at different temperature gradients.

Further, the PNIPAAm includes at least two phases of solid and liquid.

Further, the PNIPAAm is in the solid state of milky gel at 37°C, and in the liquid state of transparent aqueous solution at room temperature of 25°C.

Further, the PNIPAAm is used as a support matrix for stem cell growth in solid state, and used for collection and separation in liquid state.

The poly N isopropylacrylamide (PNIPAAm) is a temperature-sensitive hydrogel, which can sense small changes in the external environment and has good biocompatibility. When the ambient temperature is higher than the lowest critical temperature (LCST), its The volume will suddenly shrink sharply. At this time, the surface is hydrophobic, and the cells can adhere and grow. When the cells grow to a certain density, lower the temperature to LCST to make the surface of the hydrogel hydrophilic, so that the cells will detach from the hydrogel. Instead of traditional enzyme digestion, the structure and function of the cell surface are well maintained, and the tedious operation of enzymatic digestion of cells is avoided, which greatly reduces the workload.

The macromolecular chain of poly-N isopropylacrylamide (PNIPAAm) has both hydrophilic amido group and hydrophobic isopropyl group. The aqueous solution has a minimum critical temperature (LCST) of 32°C and can be cross-linked into water at 37°C. Gel is a linear aqueous solution at room temperature. Using the property of PNIPAAm to undergo a reversible phase transition near the LCST, PNIPAAm can be designed as a molecular switch, combined with stem cell culture, and the temperature sensitivity of poly-N-isopropylacrylamide (PNIPAAm) can be used to make cells stick when they are above the critical temperature. It grows on the surface of the material, and the cells detach when the temperature is below the critical temperature, which changes the traditional method of enzymatic detachment of cells. And the cells are in the three-dimensional space of the hydrogel, the cell growth state is good and the state culture efficiency is increased.

Further, the cultivation process includes the following steps:

1. Special culture medium for stem cells to cultivate stem cells.

Weigh a certain amount of poly-N-isopropylacrylamide powder into a 15ml centrifuge tube, and add an appropriate amount of stem cell-specific culture medium to dissolve it.

2. Trypsin digestion: carefully suck off the old culture medium, wash 2-3 times with PBS, add an appropriate amount of trypsin solution, digest at 37°C for 2-3 minutes, observe the cells under a microscope, the cytoplasm of the cells retracts, and no longer connects slices, indicating that the cells are digested moderately, add an equal amount of culture medium (containing 10% fetal bovine serum) to terminate the digestion. Use a gun to blow the digested cells into a suspension, add them to a 15ml centrifuge tube, centrifuge, discard the supernatant, add culture medium to blow the cells evenly, and count.

3. Add an appropriate amount of stem cells to the prepared stem cell culture medium containing PNIPAAm for culture, and place them in a 37°C cell incubator containing 5% CO ₂ for culture. At the same time, stem cells were cultured in medium without PNIPAAm as a control.

4. Change the medium after 24 hours. Take the petri dish out of the incubator and place it at room temperature for 5-6 minutes. After the hydrogel changes from milk state to transparent water, transfer the cells to a 15ml centrifuge tube for centrifugation, discard the supernatant, and add stem cell culture The cells were evenly pipetted with liquid, then inoculated into culture dishes, and placed in a 5% CO ₂ incubator at 37°C for culture.

The beneficial effects of the present invention are:

1. PNIPAAm has good biocompatibility, and cells are harvested without enzyme digestion to avoid damage to cell surface proteins.

2. The three-dimensional culture of hydrogel increases the space for cell growth and improves the efficiency of culture.

3. Compared with traditional nanofibrous scaffolds and porous scaffolds, the cross-linked network of hydrogel scaffolds contains a lot of water, which can supply nutrients to cells well, and can also cross-link bioactive factors to regulate cell growth and differentiation, so The hydrogel scaffold can better simulate the tissue-like physical and spatial structure required for cell growth, and has high plasticity, relatively simple manufacturing process, and convenient clinical application

4. During routine culture in an incubator at 37°C, because the temperature is lower than the critical dissolution temperature, the PNIPAAm on the surface of the culture dish is in a state of molecular contraction, which does not affect the normal adherent growth of cells; at about 25°C, due to the water absorption of PNIPAAm molecules Swelling, the adsorption force to the cells weakens, prompting the cells to fall off.

Detailed ways

The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the specific embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by persons of ordinary skill in the art without creative efforts fall within the protection scope of the present invention.

Embodiment 1, human umbilical cord mesenchymal stem cell culture

Instrument: confocal microscope

Main reagent: poly N-isopropylacrylamide (PNIPAAm), purchased from sigma company

Material: human umbilical cord mesenchymal stem cells

1. Human umbilical cord mesenchymal stem cells are cultivated to 80% in special culture medium for mesenchymal stem cells.

Weigh a certain amount of poly-N-isopropylacrylamide powder into a 15ml centrifuge tube, add an appropriate amount of special culture medium for mesenchymal stem cells to dissolve it. (Generally, 2g of poly-N-isopropylacrylamide is added to 10ml of culture medium, and there are slight differences between different cells)

Human umbilical cord mesenchymal stem cell culture medium preparation: 10%FBS+2%L-Glutamine+DMEM-F12+penicillin concentration is 100u/ml+streptomycin solution concentration is 100ug/ml

2. Trypsin digestion: carefully suck off the old culture medium, wash 2-3 times with PBS, add an appropriate amount of trypsin solution (1ml trypsin in a 10cm culture dish), digest at 37°C for 2-3 minutes, and observe the cells under a microscope. The cytoplasm is retracted and no longer connected into pieces, indicating that the cells are digested moderately. Add an equal amount of culture medium (containing 10% fetal bovine serum) to stop the digestion. Use a gun to blow the digested cells into a suspension, add it to a 15ml centrifuge tube at a speed of 1000 rpm, centrifuge for 4 minutes, discard the supernatant, add the special culture medium containing PNIPAAm mesenchymal stem cells, and blow the cells evenly ,count.

3. Add 3ml of prepared special culture medium for mesenchymal stem cells (including PNIPAAm) to the 6-well culture dish, the number of cells is 1-5×10 ⁵ /ml, and place in a 37°C 5% CO ₂ incubator for culture. At the same time, the mesenchymal stem cells were cultured with the special culture medium for mesenchymal stem cells not containing PNIPAAm as a control.

4. After 24 hours, replace the medium with preheated culture medium. Take the 6-well plate culture dish out of the incubator and place it at room temperature for 5-6 minutes. After the hydrogel completely changes from milk state to transparent water state, transfer the cells to a 15ml centrifuge tube and centrifuge at 1000 rpm. , centrifuged for 4 minutes, discarded the supernatant, added 1ml of special culture medium for mesenchymal stem cells and pipet the cells evenly, seeded human umbilical cord mesenchymal stem cells in a 6-well culture dish, added 3ml of prepared mesenchymal stem cell special The culture medium (including PNIPAAm) was cultured in a 37°C incubator containing 5% CO ₂ .

5. After five days of cell counting observation, it was found that the number of cells added with PNIPAAm was nearly 2 times that of cells without PNIPAAm.

The above descriptions are only preferred embodiments of the present invention, and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included in the scope of the present invention. within the scope of protection.

Claims (8)

1. a method for stem cell large scale culturing, is characterized in that: have employed PNIPAAm in culturing process.

2. the method for stem cell large scale culturing as described in the appended claim 1, is characterized in that: described PNIPAAm in culturing process as the carrier matrix of stem cell growth.

3. the method for stem cell large scale culturing as described in claim 1 or 2: it is characterized in that: in culturing process, arrange different thermogrades, described PNIPAAm is in different phases in different thermogrades.

4. the method for stem cell large scale culturing as claimed in claim 3, is characterized in that: described PNIPAAm at least comprises solid-state and liquid two kinds of phases.

5. the method for stem cell large scale culturing as claimed in claim 3, is characterized in that: described PNIPAAm is solid-state 37 DEG C time, is liquid during room temperature 25 DEG C.

6. the method for stem cell large scale culturing as described in claim 4 or 5, is characterized in that: described PNIPAAm as the support matrix of stem cell growth, is used for time liquid collecting and being separated when solid-state.

7. the method for stem cell large scale culturing as recited in claim 6, is characterized in that, comprise the following steps:

A, stem cell special culture solution culturing stem cells;

B, tryptic digestion:

C, proper amount of dry cell is added the nutrient solution containing PNIPAAm prepared cultivate.

8. the method for stem cell large scale culturing as recited in claim 6, it is characterized in that: in step c, the envrionment temperature of cultivation is 37 DEG C, and containing 5%CO ₂cell culture incubator is cultivated, and changes liquid after 24 hours.