CN113430164A

CN113430164A - Cell culture system, culture method and application

Info

Publication number: CN113430164A
Application number: CN202110596156.0A
Authority: CN
Inventors: 胡豫; 梅恒; 姚惟琦; 唐宇翔
Original assignee: Tongji Medical College of Huazhong University of Science and Technology
Current assignee: Wuhan Jimeini Technology Co.,Ltd.
Priority date: 2021-05-30
Filing date: 2021-05-30
Publication date: 2021-09-24
Anticipated expiration: 2041-05-30
Also published as: CN113430164B

Abstract

The invention discloses a cell culture system, a culture method and application, wherein the culture system comprises fibrinogen and thrombin, the concentration of the fibrinogen is 0.4-0.6 mg/ml, and the concentration of the thrombin is 4 x 10 < -3 > to 6 x 10 < -3 > U/mul. The cell culture system provided by the invention is suitable for mesenchymal stem cells, not only can keep the high differentiation capacity of the cells, but also can keep the high cell activity, and maintain the mechanical property and good shape of the cell surface.

Description

Cell culture system, culture method and application

Technical Field

The invention relates to the technical field of biology, in particular to a cell culture system, a culture method and application.

Background

Mesenchymal Stem Cells (MSCs) are derived from the mesoderm in the early development stage, are pluripotent Stem Cells with high self-renewal capacity, high proliferation capacity and multidirectional differentiation potential, are widely present in various tissues such as bone marrow, fat, umbilical cord and the like, can be cultured and expanded in vitro, can be differentiated into adipocytes, osteoblasts, cartilage tissues, nerve Cells, liver Cells and the like under the control of specific conditions, and have great application values in the aspects of cell therapy and tissue engineering.

There are also some studies on the function of promoting the differentiation of stem cells in the prior art, for example, CN102046188A discloses a blocking agent containing mesenchymal stem cells, but the purpose of the blocking agent is to make the cells better migrate from the culture system to the human body, and not suitable for in vitro culture.

At present, due to the potential of self-renewal and multi-directional differentiation of MSCs, MSCs are widely used in clinical trials related to various tissue and organ repairs, immune system diseases and the like, and involve mechanisms in aspects of MSC homing, cell repair, paracrine, immunoregulation and the like. The development of an in vitro rapid culture method which can increase the dryness of the MSC, change the cell membrane surface state of the MSC and improve the biological function of the MSC has important significance. For in vitro culture, a culture system capable of maintaining the cell functions of MSCs such as proliferation and differentiation is still needed, so that the MSCs can play a greater application value in cell therapy, tissue engineering and the like.

Disclosure of Invention

Technical problem to be solved

In order to overcome the defect that an in-vitro culture system for effectively enhancing and maintaining functions of umbilical cord mesenchymal stem cells is lacked in the prior art, the invention provides a cell culture system, a culture method and application.

The shape, differentiation capacity, cell activity and functional activity of the stem cells are closely related to the microenvironment in which the stem cells are located, the culture components and the concentration of the culture components of an in vitro culture system directly influence the growth microenvironment of the cells, and the change of the tiny concentration can cause great difference of the growth states of the stem cells. Particularly for three-dimensional culture, the components and concentration of the culture system not only directly form a growth microenvironment, but also relate to whether the formed microenvironment is uniform or not and whether cells can grow in a target environment or not, which also causes a difficult problem for the in vitro three-dimensional culture of the cells. The inventors of the present invention have surprisingly found through a large number of experiments that when the culture system of the present invention is used for culturing mesenchymal stem cells, the differentiation capacity of the cells can be improved, and high cell activity, cell surface mechanical properties and good morphology can be maintained. The process can be regarded as a pretreatment of the mesenchymal stem cells, and in the actual production process of the mesenchymal stem cell products (preparations or medicines), the dryness can be improved and other good characteristics can be kept through the pretreatment, so that the process is more beneficial to the development of the cell-forming products.

(II) technical scheme

In order to achieve the purpose, the invention provides the following technical scheme: there is provided a cell culture system comprising Fibrinogen (Fibrinogen) and Thrombin (Thrombin),

the concentration of the fibrinogen is 0.4-0.6 mg/ml;

the concentration of the thrombin is 4 multiplied by 10 < -3 > to 6 multiplied by 10 < -3 > U/mul.

In the present invention, the fibrinogen concentration is preferably 0.5 mg/ml.

In the present invention, the concentration of thrombin is preferably 5.67X 10-3U/. mu.l.

The cell culture system of the present invention preferably further comprises a buffer, preferably a T7 buffer, the T7 buffer preferably comprises Tris and NaCl, the concentration of Tris is preferably 50mM, the concentration of NaCl is preferably 150mM, and the pH of the T7 buffer is preferably 7.4. In a preferred embodiment, the T7 buffer consists of 50mM Tris and 150mM NaCl, pH 7.4.

The cell culture system of the present invention preferably further comprises a cell complete medium. The complete medium may be a complete medium conventionally used in the art to culture mesenchymal stem cells.

The term "cell culture system" as used herein is to be understood as an in vitro culture system capable of growing cells therein.

The cell culture system can be used for culturing umbilical cord mesenchymal stem cells, such as human umbilical cord mesenchymal stem cells, and the seeding density of the mesenchymal stem cells to the culture system is preferably 2 × 104-8 × 104 cells/ml, and is preferably 5 × 104 cells/ml.

The second technical scheme of the invention is as follows: provided is an in vitro culture method of mesenchymal stem cells, comprising the steps of:

(1) mixing fibrinogen and mesenchymal stem cells, wherein the concentration of the fibrinogen in the obtained mixed solution is 0.4-0.6 mg/ml;

(2) adding 4 x 10 < -3 > to 6 x 10 < -3 > U/. mu.l of thrombin into the mixed solution obtained in the step (1).

The concentration of fibrinogen in the mixed solution in the step (1) is preferably 0.5 mg/ml;

the concentration of thrombin in the mixture in step (2) is preferably 5.67X 10-3U/. mu.l.

Wherein, the mesenchymal stem cell is preferably umbilical cord mesenchymal stem cell, such as human umbilical cord mesenchymal stem cell.

In step (1) of the present invention, the concentration of fibrinogen is preferably adjusted by a buffer.

The buffer is preferably a T7 buffer.

The T7 buffer preferably comprises Tris, preferably at a concentration of 50mM, and NaCl, preferably at a concentration of 150mM, and the T7 buffer preferably has a pH of 7.4.

In step (1) of the present invention, the mixing is preferably performed in a volume ratio of 1:2 to 2:1, preferably 1: 1.

In the step (1), the concentration of the mesenchymal stem cells in the mixed solution is preferably 2 × 104-8 × 104 cells/ml; preferably 5X 104/ml.

The in vitro culture method of the present invention preferably further comprises the following steps:

(3) incubating the mixed solution obtained in the step (2) until gel is formed, wherein the incubation time is, for example, 10-30 min, for example, 15min, and the incubation temperature is, for example, 37 ℃;

(4) cell complete medium was added.

In the step (3), adjustment can be made according to the actual gel condition, and the time can be appropriately prolonged if no gel exists.

The third technical scheme of the invention is as follows: a composition is provided comprising the cell culture system described above and a mesenchymal stem cell.

The mesenchymal stem cells are preferably umbilical cord mesenchymal stem cells, preferably human umbilical cord mesenchymal stem cells, and the concentration of the mesenchymal stem cells is preferably 2 × 104-8 × 104 cells/ml; preferably 5X 104/ml.

The fourth technical scheme of the invention is as follows: a kit comprising the above cell culture system or composition is provided. The kit can activate the mesenchymal stem cells (enhance the dryness of the mesenchymal stem cells), and is more beneficial to developing the mesenchymal stem cells into cell products.

The fifth technical scheme of the invention is as follows: provides an application of the cell culture system, the composition or the kit in culturing human umbilical cord mesenchymal stem cells.

The mesenchymal stem cells are preferably umbilical cord mesenchymal stem cells, preferably human umbilical cord mesenchymal stem cells.

The culture is preferably an in vitro culture.

In the present invention, the concentrations are all final concentrations in the mixed system unless otherwise noted.

On the basis of the common knowledge in the field, the above preferred conditions can be combined randomly to obtain the preferred embodiments of the invention.

The reagents and starting materials used in the present invention are commercially available.

(III) advantageous effects

Compared with the prior art, the invention has the following beneficial effects:

when the mesenchymal stem cell culture system provided by the invention is used for culturing mesenchymal stem cells, the high differentiation capacity of the cells can be maintained, and the gene expression amounts of OCT4, SOX2, Nanog and actin are improved; can also keep high cell activity, maintain the mechanical property and good shape of the cell surface. When the method is used in the actual process of industrially producing mesenchymal stem cell products (preparations or medicines), the dryness can be improved and other good characteristics can be kept through the 'pretreatment' culture, and the method is more beneficial to the development of cell products.

Drawings

FIG. 1 shows the results of qPCR assays.

FIG. 2A shows the flow cytometry results of the control group.

FIG. 2B shows the results of the Fibrinogen-treated group flow cytometry assay.

FIG. 2C shows the results of the Fibrinogen + Thrombin-treated group flow cytometric assay.

FIG. 3A is an AFM image of the cell surface topography of the control group.

FIG. 3B is an AFM image of the cell surface topography of the Fibrinogen + Thrombin treated group.

FIG. 4A shows the result of the Young's modulus test of the control cells.

FIG. 4B shows the results of Young's modulus measurements on Fibrinogen + Thrombin treated cells.

FIG. 5A shows the cell surface morphology of the control group.

FIG. 5B shows the cell surface morphology of the Fibrinogen-treated group.

FIG. 5C shows cell surface morphology of Fibrinogen + Thrombin treated group.

FIG. 5D shows cell surface morphology of Fibrinogen + Thrombin treated group.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example 1 cell culture

Reagent material

T7 buffer configuration (pH7.4, 50mM Tris, 150mM NaCl) specific formulation (605.7mg Tris, 876.6mg NaCl, 100mL water, pH adjusted to 7.4)

Fibrinogen Fibrinogen (sigma)

Thrombin (sigma)

Procedure for the preparation of the

(1) Adjusting the concentration of hUC-MSCs (derived from human umbilical cord) to 1 × 105 cells/ml, and diluting Fibrinogen to target concentration (1mg/ml and 2mg/ml) by adopting a T7 buffer solution to prepare a solution A;

(2) mixing the MSC cell suspension with the solution A according to the ratio of 1: 1;

(3) adding 15 μ l of thrombin (initial concentration of 0.1U/μ l, final concentration of 5.67X 10-3U/μ l) into 250 μ l of MSC/A mixed solution, and stirring with a sterilized gun head during the addition;

(4) the mixture was blown up and mixed, incubated at 37 ℃ for 15min (adjusted according to the actual gel conditions without appropriate extension of the gel) to form a gel, and 1ml of complete cell culture medium (90% DMEM/F12(BI) and 10% fetal bovine serum (BI)) pre-warmed to 37 ℃ was added.

(5) Placing the mixture in a cell culture box for culture.

Example 2 qPCR assay

Experimental setup

1. Control group: fibrinogen and thrombin are not added;

MSC 1.0: the concentration of fibrinogen in the mixed solution is 0.5mg/ml, and the concentration of thrombin is 5.67 multiplied by 10 < -3 > U/[ mu ] l;

MSC 2.0: the fibrinogen concentration in the mixture was 1mg/ml and thrombin was 5.67X 10-3U/. mu.l.

1 Primary reagent Material

Total RNA extraction kit (tiangen): a total RNA extraction kit (DP430) for cultured cells/bacteria;

RNA reverse transcription kit (nunoprazan): HiScript II Q RT Supermix for qPCR (+ gDNA wiper) (R223-01);

qPCR kit (nunoprazan): AceQ qPCR SYBR Green Master Mix (Q111-02).

2 method of experiment

2.1 RNA extraction

The drug-treated cells were centrifuged to remove the supernatant, and approximately 5X 105 to 1X 106 cells were taken for RNA extraction.

An enzyme-free tip, a centrifuge tube, and the like were prepared, and RNA extraction was performed using a cultured cell/bacteria total RNA extraction kit (DP430) from Tiangen corporation.

The RNA bands were detected by electrophoresis on a 2% agarose gel and the concentration of RNA was determined using Nanodrop lite.

2.2 RNA reverse transcription

Based on the RNA concentrations determined in the above procedure, all samples were subjected to RNA reverse transcription in a total amount of 1 ug.

Reverse transcription was performed using HiScript II Q RT Supermix for qPCR (+ gDNA wiper) (R223-01) kit from Novozan.

2.3 qPCR assay

The AceQ qPCR SYBR Green Master Mix (Q111-02) kit of Novozan company is used for qPCR reaction, and the used fluorescence qPCR instrument is an FQD-96A PCR detection system of Hangzhou Bori science and technology ltd (BIOER).

The cDNA loading was 2 ul. For each sample 3 replicates per index were made.

The internal reference was human GAPDH and the qPCR primer sequences (5 '-3') for each index are shown in table 1:

TABLE 1

3. Results

As shown in FIG. 1, the human umbilical cord mesenchymal stem cells cultured by the culture system of the invention all have increased expression levels of OCT4, SOX2 and Nanog, and have decreased expression level of actin.

OCT4 has an extremely important role in stem cell pluripotency and self-renewal, SOX2 is a necessary condition for stem cell self-renewal and pluripotency, and Nanog is thought to play an important role in regulating stem cell proliferation, renewal, and pluripotency. Actin is the maintenance of cytoskeletal integrity.

Experimental setup

1. Control group: fibrinogen and thrombin are not added;

fibrinogen treatment group (Fibrinogen concentration 0.5mg/ml in the mixture);

fibrinogen + Thrombin (Fibrinogen concentration in the mixture 0.5mg/ml, Thrombin 5.67X 10-3U/. mu.l).

The microphotographs of the cultured cells of each group are shown in FIGS. 5A to D, respectively.

Example 3 cell viability assay

1 laboratory apparatus

Cytometric instrument/manufacturer: countstar/model: countstar Rigel S5

2 Primary reagent

AO/PI staining solution

3 Experimental methods

Diluting the cell suspension to a certain concentration, preparing 10 mul of cell suspension to be detected, adding the cell suspension into a 1.5ml EP tube, adding 10 mul of AO/PI dye solution, and uniformly mixing;

adding 20 mul of the mixed cell suspension into a sample adding hole of a cell counting plate, and then putting the cell suspension into a detection port;

entering a computer control system, counting, finding 3 areas for measurement, and taking an average value as the cell concentration;

from the data list, the relevant cell concentration, viability, clumping rate, growth curve, etc. can be obtained.

4 results

As shown in table 2, there was no difference between the groups of cell viability assays, indicating that the mesenchymal stem cells cultured by this method did not affect the normal growth of the cells.

TABLE 2

Example 4 flow cytometry detection of Stem cell phenotype

1 laboratory apparatus

Flow cytometer/manufacturer: beckman/model: cytoflex s

2 Experimental reagent

Mesenchymal stem cell phenotype detection kit: QB Human MSC Analysis Kit (Beijing Kuangbo)

3 Experimental methods

A three-tube protocol is typically employed for each sample;

cells were collected and washed once with pre-chilled PBS;

three 12X 75mm flow tubes are used for each sample, the control, the positive and the negative are respectively marked, and 100 mul of sample is added into each tube;

adding 20 mul of isotype control tube reagent into a control tube, adding 20 mul of positive index tube reagent into a positive tube, and adding 20 mul of negative index tube reagent into a negative tube;

mixing, incubating at room temperature (20-25 deg.C) for 20-30 min;

adding 1-2ml buffer solution, centrifuging for 5min at 300g, and removing supernatant;

adding 0.3ml of cell staining buffer solution into each tube and mixing uniformly;

and finishing the sample processing. The flow cytometry analysis can be carried out, the sample is required to be uniformly mixed before the sample is loaded, and the dyed sample is required to avoid illumination.

4 results

As shown in fig. 2A-2C, there was no difference between the groups, indicating that 3D culture of cells did not have the essential characteristics that affect the health that the cells should have.

Example 5 atomic mechanical testing

1 laboratory apparatus

Atomic mechanical microscope AFM (Asylum Research Cypher S AFM);

AC160TS-R3 tip(Olympus)

2AFM Structure

The AFM mainly comprises a laser unit, a micro-cantilever unit, a piezoelectric scanning unit and a photoelectric detection and feedback unit.

2.1 micro-cantilevers

The micro-cantilever has two types of rectangle and triangle, the force elastic constant of the micro-cantilever is between 0.1-lOON/m, even if the acting force of a few tenths of nano newtons (nN) between the probe and the sample can be easily detected. The resonance frequency of the microcantilever is typically greater than 10kHz in order to reduce vibration and acoustic wave interference. The mass of the micro-cantilever tends to decrease more and less because the resonance frequency decreases as the force spring constant decreases while the resonance frequency remains constant.

2.2 Probe

The AFM probe is typically single crystal silicon (Si) and silicon nitride (Si N) and the probe tip is in the shape of a square pyramid, a cone or a super tip. The radius of curvature of the pyramidal probe is 5-10 nm, and the radius of curvature of the silicon nitride is 20-60 nm. The super AFM probe is formed by adhering a carbon nano tube on the original AFM tip, the curvature radius is 0.5-2 nm, and the resolution of an image is higher. When the sample size is comparable to or smaller than the radius of curvature of the tip, a "broadening effect" occurs, affecting the accuracy of the image. The more slender the tip is, the higher the quality of the scanned image, and the closer the vacuum topography of the sample surface is.

3 cell processing procedure

Three groups of cells were cultured in a petri dish for 36h with a 35mm polyethylene petri dish to which a 22X 22mm silica cover plate was added, to give a final cell density of 70%. After the cell culture was completed, the cells were washed 3 times with 1mL of phosphate buffer, and fresh medium for the next experiment (nanoindentation) was added.

4 sample preparation

Transferring cultured cells on a glass sheet, then attaching the lower surface of the glass sheet to a silicon sheet substrate of a sample table, and sucking liquid on the periphery of the culture medium on the upper surface by using a liquid sucking film to keep the surface of the sample dry as much as possible. Further vacuum drying is required because the liquid on the surface of the sample easily affects the degree of vacuum of the apparatus and the accuracy of the test.

5 test procedure

(1) Starting an AFM instrument: the connection line between the power supply and the control cabinet is correct and does not need to be confirmed. And sequentially turning on a computer power supply, a case low-voltage power supply, a high-voltage power supply, a laser power supply and the like.

(2) Sample-probe feed: sample feed provides both coarse and fine feed mechanisms. The sample is fed roughly to about 1mm from the probe, and then the fine adjustment mechanism is used to slowly feed the sample while observing the change of the light spot at the PSD position, wherein the indication is that the sample is close to entering a feedback state, and then the feedback signal is observed to more slowly feed the sample until the PSD signal shows about 1.600 and the Z feedback signal is about-200 to-300. Note that the coarse and fine feed mechanisms cannot be moved any more. The following operations then allow the computer control system to automatically adjust and maintain the spacing between the sample and the probe.

(3) Sample scanning: and running a computer scanning program, and setting scanning parameters as required. The single scanning time is set to about 15s, and the scanning working state is entered.

(4) Image display and storage: the scanning process is automated and the image is displayed in a line-by-line display. The scanning is circularly repeated in the same area without changing the scanning parameters. The scanning area and the scanning range can also be changed as required. For satisfactory images, the image can be captured and stored at any time. When storing, the computer automatically saves the image information and the scanning parameter information.

(5) Exiting the scanning: if the desired image has been acquired, no further scanning is performed, the "exit" key is pressed to exit the scanning sequence, the sample is slowly moved back with fine adjustment until the Z feedback signal is above 500, and the fine feed mechanism is moved back to the bottom, and coarse movement is performed.

(6) And (3) shutting down an AFM instrument: and when the sample scanning experiment is not carried out any more, the laser power supply, the high-voltage power supply, the case low-voltage power supply and the like are sequentially turned off.

6 data processing and Young modulus calculation process

(the test was performed by Wuhan Qijing Biotech Co., Ltd.)

The specific calculation formula is as above: coeffient values + -one standard definition

a＝6.0571e+010±2.9e+010

b＝-7.6742e-007±1.22e-009

c-2.4374 e-007 ± 1.83e-007Coefficient List in the txt format in the folder, on the basis of which the young's modulus is multiplied by a factor of 7.45 in Pa.

7 results of atomic mechanics

The morphology detection is shown in fig. 3A and 3B, and the mechanical detection result is shown in fig. 4A and 4B, which shows that the cells become soft and the surface is smoother after 3D culture, and is more beneficial to biological performance.

It is noted that, herein, relational terms such as first and second, and the like may be used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Also, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus. Without further limitation, an element defined by the phrase "comprising an … …" does not exclude the presence of other identical elements in a process, method, article, or apparatus that comprises the element.

Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.

Claims

1. A cell culture system comprising fibrinogen and thrombin,

the concentration of the fibrinogen is 0.4-0.6 mg/ml,

2. The cell culture system of claim 1, further comprising a cell completion medium;

and/or, the fibrinogen concentration is 0.5 mg/ml;

and/or the concentration of thrombin is 5.67X 10-3U/. mu.l.

3. The cell culture system of claim 1, further comprising a buffer;

the buffer solution is T7 buffer solution; the T7 buffer comprises Tris and NaCl, the concentration of the Tris is 50mM, the concentration of the NaCl is 150mM, and the pH value of the T7 buffer is 7.4.

4. A cell culture method is characterized in that the method specifically comprises the following steps:

(2) adding 4 x 10 < -3 > to 6 x 10 < -3 > U/. mu.l of thrombin into the mixed solution obtained in the step (1),

the concentration of fibrinogen in the mixed solution in the step (1) is 0.5 mg/ml;

the concentration of the thrombin in the mixed solution in the step (2) is 5.67 multiplied by 10 < -3 > U/. mu.l.

5. The cell culture method according to claim 4, wherein in step (1), the concentration of fibrinogen is adjusted by a buffer before mixing, the buffer is a T7 buffer, the T7 buffer comprises Tris at a concentration of 50mM and NaCl at a concentration of 150mM, and the T7 buffer has a pH of 7.4;

and/or, mixing in the step (1) according to the volume ratio of the fibrinogen to the mesenchymal stem cells of 1:2 to 2:1, wherein the mixing is 1: 1.

6. The cell culture method according to claim 4, wherein the concentration of mesenchymal stem cells in the mixed solution in step (1) is 2X 104 to 8X 104 cells/ml; 5X 104/ml;

and/or, the mesenchymal stem cell is an umbilical cord mesenchymal stem cell.

7. The cell culture method of claim 4, further comprising the steps of:

(4) cell complete medium was added.

8. A composition comprising the cell culture system of any one of claims 1-3 and mesenchymal stem cells;

the mesenchymal stem cells are umbilical cord mesenchymal stem cells, and/or the concentration of the mesenchymal stem cells is 2 x 104-8 x 104 cells/ml; 5X 104/ml.

9. A kit comprising a cell culture system according to any one of claims 1 to 3, or a composition according to claim 8.

10. Use of the cell culture system of any one of claims 1 to 3, the composition of claim 8, or the kit of claim 9 in culturing mesenchymal stem cells;

the mesenchymal stem cells are umbilical cord mesenchymal stem cells;

and/or, the culture method is in vitro culture.