CN114480268B - Preparation method of human umbilical cord mesenchymal stem cells - Google Patents
Preparation method of human umbilical cord mesenchymal stem cells Download PDFInfo
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- CN114480268B CN114480268B CN202210076622.7A CN202210076622A CN114480268B CN 114480268 B CN114480268 B CN 114480268B CN 202210076622 A CN202210076622 A CN 202210076622A CN 114480268 B CN114480268 B CN 114480268B
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- C12N5/0602—Vertebrate cells
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
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Abstract
The invention discloses a preparation method of human umbilical cord mesenchymal stem cells, which comprises the following steps: s10, stripping Wharton' S jelly from an umbilical cord; s20, shearing the Whatman gum, adding physiological saline, uniformly mixing, centrifuging, and removing the supernatant to obtain a tissue block; s30, mixing the tissue blocks with a complete culture medium to obtain tissue block liquid; s40, inoculating the tissue block liquid into a T75 culture bottle, adding a complete culture medium, culturing until the cell fusion degree is 85%, washing, digesting, stopping digestion, and collecting cells after the digestion is stopped to obtain P0 generation cells; s50, inoculating the P0 generation cells into a T175 culture flask, adding a complete culture medium, and continuing to culture to obtain the human umbilical cord mesenchymal stem cells. Firstly, stripping Wharton's jelly from an umbilical cord, shearing, centrifuging to obtain a tissue block, adding a complete culture medium to prepare a tissue block liquid, then, primarily culturing the tissue block liquid until the cell fusion degree is 85%, harvesting P0 generation cells, and finally, subculturing the P0 generation cells to obtain the human umbilical cord mesenchymal stem cells.
Description
Technical Field
The invention relates to the technical field of cell culture, in particular to a preparation method of human umbilical cord mesenchymal stem cells.
Background
The human umbilical cord mesenchymal stem cells have higher differentiation potential and can differentiate towards multiple directions. It has wide clinical application prospect in the aspects of tissue engineering such as bones, cartilages, muscles, nerves, livers, cardiac muscles and the like. For convenience of application, the human umbilical cord mesenchymal stem cells need to be prepared and cultured in vitro. Thus, there is a need for an efficient method for preparing human umbilical cord mesenchymal stem cells.
Disclosure of Invention
The invention mainly aims to provide a preparation method of human umbilical cord mesenchymal stem cells, and aims to prepare the human umbilical cord mesenchymal stem cells by the preparation method of the human umbilical cord mesenchymal stem cells.
In order to achieve the above purpose, the invention provides a preparation method of human umbilical cord mesenchymal stem cells, comprising the following steps:
s10, stripping Wharton' S jelly from an umbilical cord;
s20, shearing the Whatman gum, adding physiological saline, uniformly mixing, centrifuging, and removing the supernatant to obtain a tissue block;
s30, mixing the tissue blocks with a complete culture medium to obtain tissue block liquid;
s40, inoculating the tissue block liquid into a T75 culture bottle, adding a complete culture medium, culturing until the cell fusion degree is 85%, washing, digesting, stopping digestion, and collecting cells after the digestion is stopped to obtain P0 generation cells;
s50, inoculating the P0 generation cells into a T175 culture flask, adding a complete culture medium, and continuing to culture to obtain the human umbilical cord mesenchymal stem cells.
Optionally, the tissue mass solution has a concentration of 1.0g/ml.
Optionally, in step S40, 0.5-1.0 ml of the tissue mass solution is inoculated into a T75 culture flask.
Alternatively, in step S40, 1.0ml of the tissue mass solution is inoculated into a T75 flask.
Optionally, in step S40, 5-15 ml of complete medium is added.
Optionally, in step S40, digestion is performed using a pancreatin solution, wherein the concentration of pancreatin in the pancreatin solution is 0.625-2.5 g/L.
Optionally, in step S50, the inoculation density is 3000-7000/cm 2 。
Alternatively, in step S50, 16 to 24ml of complete medium is added.
Optionally, after step S50, the method further includes:
digesting the human umbilical cord mesenchymal stem cells by using a pancreatin solution to obtain digested human umbilical cord mesenchymal stem cells, wherein the concentration of pancreatin in the pancreatin solution is 0.625-2.5 g/L.
Optionally, in step S30, step S40 and step S50, the complete medium comprises the following components:
transforming growth factor-beta 1 Basic fibroblast growth factor, MEK inhibitor PD0325901, GSK-3 inhibitor Laduviglusib, fisetin, ALK-5 inhibitor A83-01, piperlonguminmide, dasatinib, BCL-XL inhibitor A1331852, interleukin-1, interleukin-6, sodium bicarbonate, cuSO 4 、ZnSO 4 、Na 2 SeO 3 、FeCl 3 、Na 2 SiO 3 、MnSO 4 、NH 4 VO 3 、NiSO 4 、SnCl 2 、AlCl 3 、AgNO 3 、Ba(C 2 H 3 O 2 ) 2 、KBr、CdCl 2 、CoCl 2 、CrCl 3 NaF and GeO 2 。
According to the technical scheme, wharton's jelly is firstly peeled from an umbilical cord, then sheared and centrifuged to obtain tissue blocks, a complete culture medium is added to prepare tissue block liquid, the tissue block liquid is subjected to primary culture until the cell fusion degree is 85%, P0 generation cells are harvested, and finally the P0 generation cells are subjected to subculture to obtain the human umbilical cord mesenchymal stem cells.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required in the embodiments or the description of the prior art will be briefly described, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and other related drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.
Fig. 1 is a schematic flow chart of an embodiment of a method for preparing human umbilical cord mesenchymal stem cells according to the present invention.
The achievement of the objects, functional features and advantages of the present invention will be further described with reference to the accompanying drawings, in conjunction with the embodiments.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below. It will be apparent that the described embodiments are only some, but not all, embodiments of the invention.
The specific conditions were not specified in the examples, and the examples were conducted under the conventional conditions or the conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention. In addition, the meaning of "and/or" as it appears throughout includes three parallel schemes, for example "A and/or B", including the A scheme, or the B scheme, or the scheme where A and B are satisfied simultaneously. In addition, the technical solutions of the embodiments may be combined with each other, but it is necessary to base that the technical solutions can be realized by those skilled in the art, and when the technical solutions are contradictory or cannot be realized, the combination of the technical solutions should be regarded as not exist and not within the protection scope of the present invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The human umbilical cord mesenchymal stem cells obtained by the preparation method adopted at present have the advantages of less viable cell number and low cell viability.
In view of the above, the present invention provides a method for preparing human umbilical cord mesenchymal stem cells, which is used for increasing the number of living cells and the cell viability of the prepared human umbilical cord mesenchymal stem cells. FIG. 1 is a schematic diagram showing an embodiment of a method for preparing umbilical cord mesenchymal stem cells according to the present invention.
Referring to fig. 1, in an embodiment of the invention, the preparation method of human umbilical cord mesenchymal stem cells comprises the following steps:
s10, stripping the Wharton' S jelly from the umbilical cord.
The Wharton's jelly is a gel substance constituting an umbilical cord, wherein umbilical cord mesenchymal stem cells can be isolated, and human umbilical cord mesenchymal stem cells for application can be obtained by culturing stem cell tissues in the Wharton's jelly. The specific steps of stripping the Wharton's jelly from the umbilical cord are as follows: washing the umbilical cord with 0.9% physiological saline to remove blood from the umbilical cord; then soaking the cleaned umbilical cord in 75% alcohol for 1min, and then putting into 0.9% physiological saline for cleaning; finally cutting into 3-5 cm length, putting into 0.9% physiological saline for cleaning, and obtaining the cut and cleaned umbilical cord; and stripping the Wharton's jelly in the umbilical cord after the shearing and cleaning by using tissue forceps, putting the Wharton's jelly into 0.9% physiological saline, cleaning and tearing the Wharton's jelly into thin strips, and taking the thin strip-shaped Wharton's jelly as the Wharton's jelly for subsequent use. Of course, other methods of removing Wharton's jelly from the umbilical cord may be used, as would be conventional to those skilled in the art, and the invention is not limited in this regard.
S20, shearing the Whatman gum, adding physiological saline, uniformly mixing, centrifuging, and removing the supernatant to obtain the tissue block.
Specifically, the Whatman's jelly is put into a 50ml centrifuge tube, sheared into tissue homogenate blocks with the size of 1-4 mm, added with 0.9% physiological saline until the total volume of the liquid is 45ml, evenly mixed, centrifuged for 5min under the centrifugal force of 600g, and the supernatant is removed to obtain the tissue blocks. Thus, the impurities in the Chinese trabecular gum are effectively removed through shearing, uniform mixing and centrifugation.
S30, mixing the tissue block with the complete culture medium to obtain tissue block liquid.
The concentration of the tissue mass in the obtained tissue mass solution is suitable for inoculation and culture. The concentration of the tissue block in the tissue block liquid is 1.0g/ml.
S40, inoculating the tissue block liquid into a T75 culture bottle, adding a complete culture medium, culturing until the cell fusion degree is 85%, washing, digesting, stopping digestion, and collecting cells after the digestion is stopped to obtain P0 generation cells.
In step S40, 0.5-1.0 ml of the tissue block solution is inoculated into a T75 culture flask. Preferably, in step S40, 1.0ml of the tissue mass solution is inoculated into a T75 flask, and the number of living cells obtained from P0 generation cells is higher and the cell viability is also higher.
In step S40, 5 to 15ml, such as 5ml, 10ml or 15ml, of the complete medium is added, and at this volume, the number of living cells of the harvested P0 generation cells is further increased, preferably 10ml or 15ml of the complete medium is added.
In step S40, the cells are digested with a pancreatin solution in which the concentration of pancreatin is 0.625 to 2.5g/L, such as 0.625g/L, 1.25g/L or 2.5g/L, and at which the digestion time is short, the cell viability of the harvested P0 generation cells is high, preferably the concentration of pancreatin is 2.5g/L, that is, 0.25% pancreatin. The solvent in the pancreatin solution is a conventional solvent such as PBS (phosphate buffer saline phosphate buffer salt solution), and will not be described in detail herein.
In step S40, the T75 flask after the complete medium was added was placed in CO with a volume fraction of 5% 2 Tissue mass culture was performed in an incubator at 37 ℃. After culturing for 5 to 6 days, the first liquid change (tilting T75 flask, sucking all the liquid with a 5ml pipette, and adding 5 to 15ml complete medium); after further culturing for 4 days, the second liquid change (tilting T75 flask, sucking all the liquid with a 5ml pipette, and adding 5-15 ml complete medium); the cells were then observed under a microscope and, if the cells were yellowish, the whole cell was changed.
Washing, digestion, and terminating digestion may be performed as follows: the whole culture broth was pipetted off with a 5ml pipette, washed with 10ml of 0.9% physiological saline, digested with 3ml of pancreatin solution after washing, and stopped with 10ml of complete medium after 2min of digestion. Wherein, after adding pancreatin solution, the T75 culture flask can be gently shaken, so that pancreatin can rapidly cover the bottom of the culture flask.
Treatment of residual cells in T75 flasks can be performed as follows: sucking the cell suspension remained in the T75 culture flask by using a 5ml pipette, and filtering the cell suspension by using a 100 mu m cell filter screen to obtain a first filtrate; washing the bottom of the T75 culture flask with 10-15 ml of 0.9% physiological saline to obtain a washing liquid, and filtering the washing liquid with a 100 mu m cell filter screen to obtain a second filtrate; adding the first filtrate and the second filtrate into a 50ml centrifuge tube, centrifuging for 10min under the centrifugal force of 300g, and removing the supernatant to obtain the required residual cells.
S50, inoculating the P0 generation cells into a T175 culture flask, adding a complete culture medium, and continuing to culture to obtain the human umbilical cord mesenchymal stem cells.
Since the P0 generation cells (cells after termination of digestion) are in a precipitate state, in order to facilitate the inoculation of the P0 generation cells, the P0 generation cells need to be resuspended and counted before the inoculation of the P0 generation cells, specifically, 5 to 10ml of 0.9% physiological saline is added to the P0 generation cells, and the cells are uniformly blown to obtain a cell suspension and counted. At the time of inoculation, 1×10 6 The inoculum size of each was inoculated with P0 generation cells.
According to the technical scheme, wharton's jelly is firstly peeled from an umbilical cord, then sheared and centrifuged to obtain tissue blocks, a complete culture medium is added to prepare tissue block liquid, the tissue block liquid is subjected to primary culture until the cell fusion degree is 85%, P0 generation cells are harvested, and finally the P0 generation cells are subjected to subculture to obtain the human umbilical cord mesenchymal stem cells.
In the step S50, the inoculation density is 3000-7000/cm 2 Such as 3000 pieces/cm 2 6000 pieces/cm 2 Or 7000 pieces/cm 2 At this density, the human umbilical cord mesenchymal stem cells obtained by culture have high cell fusion degree and a large number of living cells. Preferably, the inoculation density is 6000/cm 2 。
In step S50, 16 to 24ml, such as 16ml, 22ml or 24ml, of complete medium is added, and at this volume, the number of living cells of the harvested human umbilical cord mesenchymal stem cells is further increased, preferably 22ml of complete medium is added.
After step S50, further includes: digesting the human umbilical cord mesenchymal stem cells by using a pancreatin solution to obtain digested human umbilical cord mesenchymal stem cells, wherein the concentration of pancreatin in the pancreatin solution is 0.625-2.5 g/L. When the concentration of pancreatin is more than 2.5g/L, the digestion time is too short, which is not beneficial to batch collection; when the concentration of pancreatin is less than 0.625g/L, the digestion time is too long, and the cell viability is lowered. Thus, the concentration of pancreatin in the present invention is preferably 0.625 to 2.5g/L, such as 0.625g/L, 1.25g/L or 2.5g/L, more preferably the concentration of pancreatin is 1.25g/L, i.e. 0.125% pancreatin.
In step S30, step S40 and step S50, the complete medium comprises the following components: transforming growth factor-beta 1 Basic fibroblast growth factor, MEK inhibitor PD0325901, GSK-3 inhibitor Laduviglusib, fisetin, ALK-5 inhibitor A83-01, piperlonguminmide, dasatinib, BCL-XL inhibitor A1331852, interleukin-1, interleukin-6, sodium bicarbonate, cuSO 4 、ZnSO 4 、Na 2 SeO 3 、FeCl 3 、Na 2 SiO 3 、MnSO 4 、NH 4 VO 3 、NiSO 4 、SnCl 2 、AlCl 3 、AgNO 3 、Ba(C 2 H 3 O 2 ) 2 、KBr、CdCl 2 、CoCl 2 、CrCl 3 NaF and GeO 2 . Of course, the complete medium further comprises conventional components such as IMDM basal medium and amino acid, referring to table 1, and the complete medium is used for preparing human umbilical cord mesenchymal stem cells, so that the cell culture period is shorter, the cell culture quantity is more, and the purity is higher.
TABLE 1 Components of complete Medium
The following technical solutions of the present invention will be described in further detail with reference to specific examples and drawings, and it should be understood that the following examples are only for explaining the present invention and are not intended to limit the present invention.
Example 1
(1) The Wharton's jelly is stripped from the umbilical cord.
(2) Cutting the Whatman gum, adding normal saline, mixing, centrifuging, and removing supernatant to obtain tissue blocks.
(3) The tissue mass and the complete medium were mixed to obtain a tissue mass solution (concentration of tissue mass: 1.0 g/ml).
(4) 1.0ml of the tissue block liquid is inoculated into a T75 culture bottle, 10ml of complete culture medium is added, the culture is carried out until the cell fusion degree is 85%, the culture is washed, the digestion is stopped by using pancreatin solution (the concentration of pancreatin is 2.5 g/L), and cells after the digestion is stopped are collected, so that P0 generation cells are obtained.
(5) At 6000 pieces/cm 2 Will be 1X 10 6 And inoculating the P0 generation cells into a T175 culture flask, adding 22ml of complete culture medium, and continuing to culture to obtain the human umbilical cord mesenchymal stem cells.
(6) And digesting the human umbilical cord mesenchymal stem cells by using a pancreatin solution to obtain digested human umbilical cord mesenchymal stem cells, wherein the concentration of pancreatin in the pancreatin solution is 1.25g/L.
Wherein the complete medium in (3) - (5) comprises the components shown in Table 1.
Example 2
(1) The Wharton's jelly is stripped from the umbilical cord.
(2) Cutting the Whatman gum, adding normal saline, mixing, centrifuging, and removing supernatant to obtain tissue blocks.
(3) The tissue mass and the complete medium were mixed to obtain a tissue mass solution (concentration of tissue mass: 1.0 g/ml).
(4) Inoculating 0.5ml of the tissue block liquid into a T75 culture bottle, adding 5ml of complete culture medium, culturing until the cell fusion degree is 85%, washing, digesting with pancreatin solution (the concentration of pancreatin is 0.625 g/L), stopping digestion, and collecting cells after the digestion is stopped to obtain P0 generation cells.
(5) At 3000 pieces/cm 2 Will be 1X 10 6 And inoculating the P0 generation cells into a T175 culture flask, adding 16ml of complete culture medium, and continuing to culture to obtain the human umbilical cord mesenchymal stem cells.
(6) And digesting the human umbilical cord mesenchymal stem cells by using a pancreatin solution to obtain digested human umbilical cord mesenchymal stem cells, wherein the concentration of pancreatin in the pancreatin solution is 0.625g/L.
Wherein the complete medium in (3) - (5) comprises the components shown in Table 1.
Example 3
(1) The Wharton's jelly is stripped from the umbilical cord.
(2) Cutting the Whatman gum, adding normal saline, mixing, centrifuging, and removing supernatant to obtain tissue blocks.
(3) The tissue mass and the complete medium were mixed to obtain a tissue mass solution (concentration of tissue mass: 1.0 g/ml).
(4) Inoculating 0.8ml of the tissue block liquid into a T75 culture bottle, adding 10ml of complete culture medium, culturing until the cell fusion degree is 85%, washing, digesting with pancreatin solution (the concentration of pancreatin is 1.25 g/L), stopping digestion, and collecting cells after the digestion is stopped to obtain P0 generation cells.
(5) At 5000 pieces/cm 2 Will be 1X 10 6 And inoculating the P0 generation cells into a T175 culture flask, adding 18ml of complete culture medium, and continuing to culture to obtain the human umbilical cord mesenchymal stem cells.
(6) And digesting the human umbilical cord mesenchymal stem cells by using a pancreatin solution to obtain digested human umbilical cord mesenchymal stem cells, wherein the concentration of pancreatin in the pancreatin solution is 1.25g/L.
Wherein the complete medium in (3) - (5) comprises the components shown in Table 1.
Example 4
(1) The Wharton's jelly is stripped from the umbilical cord.
(2) Cutting the Whatman gum, adding normal saline, mixing, centrifuging, and removing supernatant to obtain tissue blocks.
(3) The tissue mass and the complete medium were mixed to obtain a tissue mass solution (concentration of tissue mass: 1.0 g/ml).
(4) 1.0ml of the tissue block liquid is inoculated into a T75 culture bottle, 15ml of complete culture medium is added, the culture is carried out until the cell fusion degree is 85%, the culture is washed, the digestion is stopped by using pancreatin solution (the concentration of pancreatin is 2.5 g/L), and cells after the digestion is stopped are collected, so that P0 generation cells are obtained.
(5) At 7000/cm 2 Will be 1X 10 6 Each said P0 generationThe cells are inoculated into a T175 culture flask, 24ml of complete culture medium is added, and the culture is continued, so that the human umbilical cord mesenchymal stem cells are obtained.
(6) And digesting the human umbilical cord mesenchymal stem cells by using a pancreatin solution to obtain digested human umbilical cord mesenchymal stem cells, wherein the concentration of pancreatin in the pancreatin solution is 2.5g/L.
Wherein the complete medium in (3) - (5) comprises the components shown in Table 1.
Example 5
(1) The Wharton's jelly is stripped from the umbilical cord.
(2) Cutting the Whatman gum, adding normal saline, mixing, centrifuging, and removing supernatant to obtain tissue blocks.
(3) The tissue mass and the complete medium were mixed to obtain a tissue mass solution (concentration of tissue mass: 1.0 g/ml).
(4) Inoculating 0.6ml of the tissue block liquid into a T75 culture bottle, adding 10ml of complete culture medium, culturing until the cell fusion degree is 85%, washing, digesting with pancreatin solution (the concentration of pancreatin is 1.25 g/L), stopping digestion, and collecting cells after the digestion is stopped to obtain P0 generation cells.
(5) At 5000 pieces/cm 2 Will be 1X 10 6 And inoculating the P0 generation cells into a T175 culture flask, adding 20ml of complete culture medium, and continuing to culture to obtain the human umbilical cord mesenchymal stem cells.
(6) And digesting the human umbilical cord mesenchymal stem cells by using a pancreatin solution to obtain digested human umbilical cord mesenchymal stem cells, wherein the concentration of pancreatin in the pancreatin solution is 1.0g/L.
Wherein the complete medium in (3) - (5) comprises the components shown in Table 1.
Example 6
The difference from example 1 is that: 15ml of complete medium was added to (4).
The number of living cells and the cell viability of the digested human umbilical cord mesenchymal stem cells obtained in examples 1 to 6 were counted, and the results are shown in Table 2.
Table 2 cases of digested human umbilical cord mesenchymal stem cells obtained in examples and comparative examples
| Number of living cells (10E 6) | Cell viability (%) | |
| Example 1 | 9.87 | 99.12 |
| Example 2 | 9.51 | 98.65 |
| Example 3 | 9.68 | 98.21 |
| Example 4 | 9.75 | 97.89 |
| Example 5 | 9.56 | 97.11 |
| Example 6 | 9.96 | 99.25 |
As can be seen from Table 2, the human umbilical cord mesenchymal stem cells obtained by the preparation method provided by the embodiment of the invention have the advantages of high number of living cells and high cell viability.
Steps (1) to (4) of example 1 were performed to obtain P0 generation cells (1.0 ml of tissue mass solution); steps (1) - (4) of example 1 were performed, but 0.5ml of the tissue mass solution was inoculated into a T75 flask in (4) to obtain P0-generation cells (0.5 ml of the tissue mass solution). The number of living cells and the cell viability of the P0 cell were counted by performing a plurality of sample tests, and the results are shown in Table 3.
TABLE 3 cases where P0 generation cells were obtained by inoculating and culturing 0.5ml and 1.0ml of tissue block solution
As can be seen from Table 3, 1.0ml of the tissue mass liquid was inoculated with a larger number of living cells than 0.5ml of the tissue mass liquid.
Steps (1) to (4) of example 1 were carried out, and the pancreatin concentration in the pancreatin solution was 2.5g/L, 1.25g/L and 0.625g/L, respectively, to obtain P0 generation cells, and the digestion time, the number of living cells of the P0 generation cells and the cell viability were counted, to obtain the results shown in table 4.
TABLE 4 cases where P0 generation cells were obtained after digestion with various pancreatin solutions
As is clear from Table 4, the digestion time of the pancreatin concentration was shorter and the cell viability was higher for 2.5g/L compared to the pancreatin concentrations of 1.25g/L and 0.625g/L.
The steps (1) to (6) of example 1 were carried out so that the pancreatin concentration of the pancreatin solution was 2.5g/L, 1.25g/L and 0.625g/L, respectively, and P2-generation human umbilical cord mesenchymal stem cells were taken and digested, and the cell digestion time of step (6), the number of living cells and the cell viability of the digested human umbilical cord mesenchymal stem cells were counted, to obtain the results shown in Table 5.
TABLE 5 digestion of P2-Generation human umbilical cord mesenchymal Stem cells with pancreatin solutions
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As can be seen from Table 5, the 2.5g/L pancreatin concentration digestion time is too short for batch collection; the digestion time of the pancreatin concentration of 0.625g/L is longer, and the cell activity is reduced due to longer digestion time; the digestion time and the cell viability of the pancreatin concentration of 1.25g/L are both better.
The inoculation densities in steps (1) to (5) and (5) of example 1 were 3000 pieces/cm, respectively 2 5000 pieces/cm 2 6000 pieces/cm 2 7000 pieces/cm 2 When the cell fusion degree of the human umbilical cord mesenchymal stem cells obtained by one inoculation density culture reaches 85%, the human umbilical cord mesenchymal stem cells obtained by each inoculation density culture are harvested, and the number of living cells and the cell viability of the human umbilical cord mesenchymal stem cells are counted, so that the results are shown in Table 6.
TABLE 6 cases of human umbilical cord mesenchymal stem cells obtained by culture at various seed densities
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As can be seen from Table 6, and3000 pieces/cm 2 6000/cm group 2 7000/cm 2 The number of living cells in the group is greater. And in the cell culture process, after each group of cells is cultured for 48 hours, the cell culture time is 3000/cm 2 Group cell fusion degree is about 20%,5000/cm 2 Group cell fusion degree is about 40%,6000/cm 2 The fusion degree of the group cells is 40-50%, 7000/cm 2 The fusion degree of the group cells is 55-60%; after culturing the cells for 72 hours (i.e., 3 days), 3000/cm 2 The fusion degree of the group cells is about 50-60%, 5000/cm 2 The fusion degree of the group cells is about 80 percent, 6000/cm 2 Group sum 7000/cm 2 The fusion degree of group cells is similar and is more than 85 percent. Description of the use of 6000/cm 2 The inoculation density of the strain is better in inoculation culture effect, the number of living cells is large, and the cell fusion degree is high.
Performing the steps (1) - (4) and (4) of example 1, and adding 10ml of complete medium to obtain P0 generation cells; steps (1) to (4) of example 1 were performed, but 5ml of complete medium was added to (4) to obtain P0 generation cells; the steps (1) - (4) and (4) of example 6 were performed, and 15ml of complete medium was added to obtain P0-generation cells. The number of living cells and the cell viability of the P0 cell were counted by performing a plurality of sample tests, and the results are shown in Table 7.
TABLE 7 case where P0-generation cells were obtained by medium culture in each culture medium
As can be seen from Table 7, the number of living cells obtained was greater in the 10ml and 15ml groups than in the 5ml group.
Carrying out the steps (1) - (5) of the example 1, and adding 22ml of complete medium to obtain human umbilical cord mesenchymal stem cells; steps (1) - (5) of example 1 were performed, but 16ml of complete medium was added to (5) to obtain human umbilical cord mesenchymal stem cells; steps (1) - (5) of example 1 were performed, but 18ml of complete medium was added to (5) to obtain human umbilical cord mesenchymal stem cells; steps (1) - (5) of example 1 were performed, but 20ml of complete medium was added to (5) to obtain human umbilical cord mesenchymal stem cells; steps (1) - (5) of example 1 were performed, but 24ml of complete medium was added to (5) to obtain human umbilical cord mesenchymal stem cells. The number of living cells and the cell viability of the human umbilical cord mesenchymal stem cells were counted by performing a plurality of sample tests, and the results are shown in Table 8.
TABLE 8 conditions of obtaining human umbilical cord mesenchymal stem cells by culture in various culture medium volumes
As can be seen from Table 8, the 22ml group obtained a greater number of living cells than the 16ml group, the 18ml group, the 20ml group and the 24ml group.
The foregoing is merely a preferred embodiment of the present invention and is not intended to limit the scope of the present invention, but various modifications and variations will be apparent to those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the scope of the present invention.
Claims (7)
1. A method for preparing human umbilical cord mesenchymal stem cells, which is characterized by comprising the following steps:
s10, stripping Wharton' S jelly from an umbilical cord;
s20, shearing the Whatman gum, adding physiological saline, uniformly mixing, centrifuging, and removing the supernatant to obtain a tissue block;
s30, mixing the tissue blocks with a complete culture medium to obtain tissue block liquid;
s40, inoculating 0.5-1 ml of the tissue block liquid into a T75 culture bottle, adding 5-15 ml of complete culture medium, culturing until the cell fusion degree is 85%, washing, digesting, stopping digestion, and collecting cells after stopping digestion to obtain P0 generation cells;
s50, inoculating the P0 generation cells into a T75 culture bottle, adding a complete culture medium, and continuously culturing to obtain human umbilical cord mesenchymal stem cells;
in step S30, step S40 and step S50, the complete medium comprises the following components:
transforming growth factor-beta 1 0.01ng/mL, 100ng/mL of basic fibroblast growth factor, 0.01 mu M, GSK-3 inhibitor Laduvigkuib 50. Mu.M of MEK inhibitor PD0325901, 100 mu M, ALK-5 inhibitor A83-01. Mu.M of fisetin, 0.1. Mu.M of piperlongum amide, 0.001 mu M, BCL-XL inhibitor A1331852 100. Mu.M of dasatinib, 1 mu.g/L of interleukin-1, 6 1 mu g/L of interleukin-and 20g/L, cuSO of sodium bicarbonate 4 20μg/L、ZnSO 4 100μg/L、Na 2 SeO 3 0.01μg/L、FeCl 3 1200μg/L、Na 2 SiO 3 100μg/L、MnSO 4 20μg/L、NH 4 VO 3 20μg/L、NiSO 4 0.01μg/L、SnCl 2 20μg/L、AlCl 3 0.01μg/L、AgNO 3 20μg/L、Ba(C 2 H 3 O 2 ) 2 0.01μg/L、KBr 20μg/L、CdCl 2 0.01μg/L、CoCl 2 20μg/L、CrCl 3 0.01 mug/L, naF mug/L and GeO 2 0.01μg/L。
2. The method for preparing human umbilical cord mesenchymal stem cells of claim 1, wherein the concentration of the tissue mass in the tissue mass solution is 1.0g/ml.
3. The method of claim 1, wherein 1.0ml of the tissue mass is inoculated into a T75 flask in step S40.
4. The method of claim 1, wherein in step S40, a pancreatin solution is used for digestion, and the concentration of pancreatin in the pancreatin solution is 0.625-2.5 g/L.
5. The method for preparing human umbilical cord mesenchymal stem cells as claimed in claim 1, wherein in step S50, the seeding is performedThe density is 3000 to 7000/cm 2 。
6. The method for preparing human umbilical cord mesenchymal stem cells according to claim 1, wherein 16-24 ml of complete medium is added in step S50.
7. The method for preparing human umbilical cord mesenchymal stem cells of claim 1, further comprising, after step S50:
and digesting the human umbilical cord mesenchymal stem cells by using a pancreatin solution to obtain digested human umbilical cord mesenchymal stem cells, wherein the concentration of pancreatin in the pancreatin solution is 0.625-2.5 g/L.
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