Rapid preparation method of umbilical cord source mesenchymal stem cells

Abstract

The invention discloses a rapid preparation method of umbilical cord-derived mesenchymal stem cells, and relates to the technical field of preparation of umbilical cord-derived mesenchymal stem cells, and the rapid preparation method comprises the following experimental steps: step 1, taking a fresh healthy umbilical cord of 16 + / -1cm, equally dividing the umbilical cord into two parts by using surgical scissors in a culture dish, respectively putting the two parts into a new culture dish, and marking the two parts as a group A; 2, 75% medical alcohol is sprayed to the umbilical cord A, and saline water is used for flushing after standing is conducted for 30 s; step 3, extracting the group A by using a straight pair of surgical forceps, a bent pair of surgical forceps, a left pair of surgical forceps and a right pair of surgical forceps, discharging blood in blood vessels in an extrusion manner, washing the blood by using saline water, putting the umbilical cord tissues into a 50ml centrifugal tube, and fully cutting the umbilical cord tissues into blocks with the size of 1.5 + / -0.1 mm by using surgical scissors for later use; according to the rapid preparation method of the umbilical cord source mesenchymal stem cells, blood vessels do not need to be removed, growth of vascular endothelial cells is prevented, and purity uniformity and biological efficacy of the mesenchymal stem cells are guaranteed to meet industrial requirements.

Description

Technical Field

[0001] The present invention relates to the technical field of umbilical cord-derived mesenchymal stem cell preparation, and in particular to a method for rapidly preparing umbilical cord-derived mesenchymal stem cells. Background Art

[0002] Umbilical cord mesenchymal stem cells (MSCs) refer to a type of multifunctional stem cells found in the umbilical cord tissue of newborns. They can differentiate into many types of tissue cells, have high differentiation potential, and can differentiate in multiple directions. They have broad clinical application prospects in tissue engineering such as bone, cartilage, muscle, tendon, ligament, nerve, liver, endothelium, and myocardium. MSCs are isolated from human umbilical cords, and their cell content and proliferation ability are better than those of bone marrow MSCs, their immunogenicity is lower than that of bone marrow MSCs, and they have the advantages of easy collection and no ethical controversy, so they are increasingly attracting the attention of researchers.

[0003] The current technology is to take a fresh healthy umbilical cord, rinse it with PBS or 0.9% saline, remove the blood vessels with scissors and forceps, peel out the Warren's jelly tissue inside, cut the obtained tissue into 1.5mm pieces, add α-MEM culture medium and place it in a 37℃, 5% CO2 incubator for culture. The culture medium contains 10% HeliosUItraGRO serum substitute. After 5-7 days of umbilical cord tissue culture, some cells can be seen crawling out from around the tissue block, and the morphology is small spindle-shaped. After a week, the cells begin to proliferate rapidly, forming cell colonies of varying sizes. After the cells are full, they are digested and amplified.

[0004] This technical method requires the removal of two arteries and one vein of the umbilical cord when the umbilical cord is crushed and extracted. This process consumes a lot of time and cost. The purpose of removing the blood vessels is to prevent the growth of endothelial cells and reduce the purity of mesenchymal stem cells. Summary of the invention

[0005] In view of the deficiencies of the prior art, the present invention provides a method for rapidly preparing umbilical cord-derived mesenchymal stem cells, which solves the problems raised in the above-mentioned background technology.

[0006] To achieve the above objectives, the present invention is implemented by the following technical scheme: A method for rapidly preparing umbilical cord-derived mesenchymal stem cells, comprising the following experimental steps:

[0007] Step 1: Take a fresh healthy umbilical cord of 16±1cm, cut it into two equal parts with surgical scissors in a culture dish, put them into new culture dishes respectively, and mark them as group A;

[0008] Step 2: Spray the umbilical cord of group A with 75% medical alcohol, let it stand for 30 seconds, and then rinse it with saline;

[0009] Step 3: For group A extraction, surgical forceps, one straight and one curved, were used in each hand to squeeze out the blood from the blood vessels and wash with saline. After the blood was fully washed, the umbilical cord tissue was placed in a 50 ml centrifuge tube and fully chopped into 1.5 ± 0.1 mm blocks using surgical scissors for standby use;

[0010] Step 4: Cut 2±0.2cm of the Pasteur pipette with surgical scissors and suck the tissues in group A into Corning 75cm 2 Cell culture bottles, 1.5 ± 0.2 ml tissue per bottle, a total of four bottles; use a 1 ml pipette to evenly distribute the tissue of group A on the bottom of the culture bottle;

[0011] Step 5: Place group A in a 37 ± 1°C, 5% CO 2 Place in the incubator for 7-9 hours;

[0012] Step 6: Prepare 60 ml of complete medium containing 10% serum replacement for standby use. Take out 30 ml of complete medium and add 1 μg rhVEGA. After 8 hours, add 10 ml of complete medium containing rhVEGA to each bottle of tissue in group A. Mix well so that the medium evenly covers the bottom of the culture bottle and place it in a 37°C, 5% CO 2 Incubator culture;

[0013] Step 7: After 90-100 hours, replace the medium with the corresponding culture medium of step 6;

[0014] Step 8: After step 7, observe the crawling out of group A cells under a microscope, and find that the crawling out ratio of group A cells is less than 20%; use the corresponding culture medium of step 6 to replace the medium;

[0015] Step 9: 72 hours after step 8, observe the crawling out of group A cells under a microscope. If the crawling out ratio of group A cells is greater than 75%, it is determined that the cells can be passaged.

[0016] Step 10: Subculture the cells of group A; tap the culture bottle lightly to remove the tissue, add 6 ml of saline and shake lightly, pour out the saline and tissue, add 2 ml of GIAICO digestion solution, observe under the microscope, after the cells become round and suspended, add 10 ml of physiological saline to dilute, and then aspirate the suspension into a 50 ml centrifuge tube; adjust the balance according to the number of tubes to collect the suspension, centrifuge at 1500 rpm for 5 minutes;

[0017] Step 11: After centrifugation, count the total number of cells in group A;

[0018] Step 12: Take 2 million cells from group A, suspend them in 50 ml of complete culture medium, and plant them in a 175 cm 2 Place the cell culture flask in 25 ml of complete culture medium and place in a 37°C, 5% CO 2 Cultured in an incubator; that is, P1 generation cells;

[0019] Step 13: After 48 hours, subculture the cells in group A; pour out the complete medium, add 6 ml of saline and shake gently to pour out evenly, add 3 ml of GIAICO digestion solution, evenly spread the bottom of the culture bottle, observe under the microscope, wait for the cells to become round and fall off, add 15 ml of physiological saline to dilute, and aspirate the suspension into a 50 ml centrifuge tube; adjust the balance according to the number of tubes to collect the suspension, and centrifuge for 5 minutes at 1500 rpm;

[0020] Step 14: Count and take out 3 million cells from group A, suspend them in 75 ml of complete medium, and plant them in three 175 cm 2 Place the cell culture flask in a 37°C, 5% CO 2 Cultured in an incubator; that is, P2 generation cells;

[0021] Step 15: By repeating the operations of step 13 and step 14, P3 generation cells, P4 generation cells, and P5 generation cells are obtained in sequence;

[0022] Step 16: After 48 hours, take a 4X photo of the cell morphology of group A; repeat step 13, collect 3 million cells from group A for flow cytometry identification, and take another 3 million cells for osteogenic, adipogenic and chondrogenic differentiation induction experiments; take another 3 million cells for soft agar clone microscopy tumorigenicity identification experiments.

[0023] Optionally, the experimental reagents and consumables are as follows:

[0024] HeliosUItraGRO serum replacement; GIBICO α-MEM medium; 10cm culture dish; Corning 75 and 175cm 2 Cell culture flask; sterile surgical scissors; tweezers; Corning 50ml centrifuge tube; 0.9% saline; 75% medical alcohol; GIBICO digestion solution; recombinant human endothelial cell inhibitory factor; THERMO centrifuge; THERMO biological safety cabinet; Pasteur pipette; 1ml and 10ml pipettes.

[0025] Optionally, in step fifteen, the acquisition interval of the P3 generation cells, the P4 generation cells, and the P5 generation cells is 48 hours.

[0026] The present invention provides a method for rapidly preparing umbilical cord-derived mesenchymal stem cells, which has the following beneficial effects:

[0027] The rapid preparation method of umbilical cord-derived mesenchymal stem cells does not require the removal of blood vessels and also prevents the growth of vascular endothelial cells, thereby ensuring that the purity uniformity and biological efficacy of the mesenchymal stem cells meet industry requirements. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 A schematic diagram of morphological comparison in the embodiment of the invention;

[0029] Figure 2 This is a flow cytometry comparison diagram of Group A1 in the embodiment of the invention;

[0030] Figure 3 This is a flow cytometry comparison diagram of Group A1 in the embodiment of the invention;

[0031] Figure 4 This is a flow cytometry comparison diagram of Group A1 in the embodiment of the invention;

[0032] Figure 5 This is a flow cytometry comparison diagram of Group A1 in the embodiment of the invention;

[0033] Figure 6 This is a flow cytometry comparison diagram of Group A1 in the embodiment of the invention;

[0034] Figure 7 This is a flow cytometry comparison diagram of Group B1 in the embodiment of the invention;

[0035] Figure 8 This is a flow cytometry comparison diagram of Group B1 in the embodiment of the invention;

[0036] Fig. 9 This is a flow cytometry comparison diagram of Group B1 in the embodiment of the invention;

[0037] Fig.10 This is a comparison diagram of adipogenesis in the differentiation induction experiment in the embodiment of the invention;

[0038] Fig.11 This is a comparison diagram of osteogenesis in the differentiation induction experiment in the embodiment of the invention;

[0039] Fig.12 This is a comparison diagram of cartilage formation in the differentiation induction experiment in the embodiment of the invention;

[0040] Fig.13 It is a negative control diagram for the soft agar clone microscopic tumorigenicity identification experiment in the embodiment of the invention;

[0041] Fig.14 It is a positive control diagram for the soft agar clone microscopic tumorigenicity identification experiment in the embodiment of the invention;

[0042] Fig.15 This is a comparison of the detection results of groups A1 and B1 in the soft agar clonal microscopic tumorigenicity identification experiment in the example of the invention. DETAILED DESCRIPTION

[0043] The technical solutions in the embodiments of the present invention will be described clearly and completely below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments.

[0044] The present invention provides a technical solution: a method for rapidly preparing umbilical cord-derived mesenchymal stem cells, comprising the following specific experimental steps:

[0045] Step 1: Take 16 cm of fresh healthy umbilical cord, cut it into two parts with surgical scissors in a culture dish, put them into new culture dishes, and mark them as group A1 and group B1 respectively;

[0046] Step 2: Spray the umbilical cords of group A1 and group B1 with 75% medical alcohol, let it stand for 30 seconds, and then rinse with saline;

[0047] Step 3: For group A1, three blood vessels were removed using surgical scissors and forceps, and the Warburg jelly tissue inside was peeled out and placed in a 50 ml centrifuge tube. The obtained tissue was cut into 1.5 ± 0.1 mm blocks using surgical scissors for standby use;

[0048] Step 4: Extraction of group B1 was performed using the technology of the present invention. One straight and one curved surgical forceps were used in each hand to squeeze out the blood from the blood vessels and wash with saline. After the blood was fully washed, the umbilical cord tissue was placed in a 50 ml centrifuge tube and fully chopped into 1.5 mm pieces using surgical scissors for standby use.

[0049] Step 5: Cut off about 2 cm of the Pasteur pipette with surgical scissors, and suck the tissues of group A1 and group B1 into Corning 75cm 2 Cell culture bottles, about 1.5 ml tissue per bottle; four bottles for group A1, four bottles for group B1 (keep the number of tissue bottles the same as group A1); use a 1 ml pipette to evenly distribute the tissues of groups A1 and B1 at the bottom of the culture bottle;

[0050] Step 6: Place groups A1 and B1 in a 37°C, 5% CO2 incubator for 8 h;

[0051] Step 7: Prepare 60 ml of complete medium containing 10% serum replacement for standby use, take out 30 ml of complete medium, add 1 μg rhVEGA, and after 8 hours, add 10 ml of complete medium to each bottle of tissue in group A1 and group B1, add ordinary complete medium to group A1; add complete medium containing rhVEGA to group B1; mix well so that the two groups of culture media evenly cover the bottom of the culture bottle, and place it in a 37°C, 5% CO2 incubator for culture;

[0052] Step 8: After 96 hours, replace the medium with the corresponding culture medium of step 7;

[0053] Step 9: 96 hours after step 8, observe the crawling out of group A1 and group B1 under the microscope, and find that a small number of cells in group A1 and group B1 crawl out; use the corresponding culture medium of step 7 to replace the medium;

[0054] Step 10: 72 hours after step 9, the crawling out of groups A1 and B1 was observed under a microscope. It was found that a large number of cells in groups A1 and B1 crawled out and could be passaged.

[0055] Step 11: Subculture the cells of group A1 and group B1; tap the culture bottle lightly to make the tissue fall off, add 6ml of saline and shake lightly, pour out the saline and tissue, add 2ml of GIB1ICO digestion solution, observe under the microscope, after the cells become round and suspended, add 10ml of physiological saline to dilute, and then aspirate the suspension into a 50ml centrifuge tube; adjust the balance according to the number of tubes to collect the suspension, centrifuge for 5min at 1500rpm;

[0056] Step 12: After centrifugation, the total number of cells in group A1 and group B1 was counted: 2.15 million cells in group A1 and 2.23 million cells in group B1;

[0057] Step 13: Take 2 million cells from each group A1 and B1, suspend them in 50 ml of complete culture medium, and plant them in two 175 cm 2 Cell culture flasks, each containing 25 ml of complete medium; placed in a 37°C, 5% CO2 incubator for culture; these are P1 generation cells;

[0058] Step 14: After 48 hours, subculture the cells of group A1 and group B1; pour out the complete culture medium, add 6 ml of saline and shake gently to pour out evenly, add 3 ml of GIBICO digestion solution, evenly spread the bottom of the culture bottle, observe under the microscope, wait for the cells to become round and fall off, add 15 ml of physiological saline to dilute, and aspirate the suspension into a 50 ml centrifuge tube; during the operation, ensure that groups A1 and B1 do not cross; balance the number of tubes for collecting suspension at 1500 rpm and centrifuge for 5 minutes;

[0059] Step 15: Count and take out 3 million cells from each of the A1 and B1 groups, suspend them in 75 ml of complete culture medium, and inoculate three 175 cm 2 The cell culture flask was placed in a 37°C, 5% CO2 incubator for culture; this is the P2 generation of cells;

[0060] Step 16: After 48 hours, repeat steps 14 and 15 to obtain P3 cells;

[0061] Step 17: After 48 hours, repeat steps 14 and 15 to obtain P4 cells;

[0062] Step 18: After 48 hours, repeat steps 14 and 15 to obtain P5 cells;

[0063] Step 19: After 48 hours, take 4X photos of the cell morphology of groups A1 and B1; repeat step 14, collect 3 million cells from groups A1 and B1 for flow cytometry identification, and take another 3 million cells for osteogenic adipogenic and chondrogenic differentiation induction experiments; and take another 3 million cells for soft agar clone microscopy tumorigenicity identification experiments.

[0064] According to the identification criteria of umbilical cord mesenchymal stem cells: (1) morphological identification; (2) flow cytometry identification (purity and homogeneity); (3) differentiation induction identification (biological efficacy experiment); (4) soft agar clone microscopy tumorigenicity identification experiment. A1 and B1 groups were compared and identified in these four aspects;

[0065] (1) Morphological comparison (anterior A1, posterior B1), such as Figure 1 As shown;

[0066] There was no obvious difference in cell shape in morphological comparison. The morphology of groups A1 and B1 both met the requirements for umbilical cord mesenchymal stem cells to adhere to the wall and were spindle-shaped;

[0067] (2) Flow cytometry comparison, such as Figures 2 to 9 As shown, flow cytometry was used to compare the results of group A1 and group B1. Although there were differences in the positive and negative indicators between the two groups, the results of the two groups met the industry standards for flow cytometry phenotype identification of human umbilical cord mesenchymal stem cells (the proportion of CD73 / CD90 / CD105 positive cells was greater than 95%; the proportion of CD11 / CD19 / CD31 / CD34 / CD45 / HLA-DR positive cells was less than 2%).

[0068] (3) Differentiation induction experiment (the order is adipogenesis, osteogenic, and chondrogenic comparison), such as Figures 10 to 12 As shown (front A1, back B1); through differentiation experiments, the A1 and B1 groups were compared, and both groups of human umbilical cord mesenchymal stem cells met the industry standards and had no difference in inducing differentiation ability;

[0069] (4) Comparison of soft agar clone microscopy tumorigenicity identification experiments, such as Figures 13 to 15 As shown (front A1, back B1), through experiments, it is known that the soft agar cloning experiments of cells in groups A1 and B1 are both negative, which meets the industry standards.

[0070] By comparing the four common standards for the identification of umbilical cord mesenchymal stem cells in the industry, it can be concluded that the existing extraction and preparation method of group A1 and the technical method of group B1 of the present invention are consistent.

[0071] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. A method for rapidly preparing umbilical cord-derived mesenchymal stem cells, characterized in that: The experimental steps include: Step 1: Take a fresh healthy umbilical cord of 16±1cm, cut it into two equal parts with surgical scissors in a culture dish, put them into new culture dishes respectively, and mark them as group A; Step 2: Spray the umbilical cord of group A with 75% medical alcohol, let it stand for 30 seconds, and then rinse it with saline; Step 3: For group A extraction, surgical forceps, one straight and one curved, were used in each hand to squeeze out the blood from the blood vessels and wash with saline. After the blood was fully washed, the umbilical cord tissue was placed in a 50 ml centrifuge tube and fully chopped into 1.5 ± 0.1 mm blocks using surgical scissors for standby use; Step 4: Cut 2±0.2cm of the Pasteur pipette with surgical scissors and suck the tissues in group A into Corning 75cm 2 Cell culture bottles, 1.5 ± 0.2 ml tissue per bottle, a total of four bottles; use a 1 ml pipette to evenly distribute the tissue of group A on the bottom of the culture bottle; Step 5: Place group A in a 37±1℃, 5% CO2 incubator for 7-9h; Step 6: Prepare 60 ml of complete medium containing 10% serum replacement for standby use, take out 30 ml of complete medium, add 1 kg of rhVEGA, and after 8 hours, add 10 ml of complete medium containing rhVEGA to each bottle of tissue in group A; mix well to make the medium evenly cover the bottom of the culture bottle, and place it in a 37°C, 5% CO2 incubator for culture; Step 7: After 90-100 hours, replace the medium with the corresponding culture medium of step 6; Step 8: After step 7, observe the crawling out of group A cells under the microscope, and find that the crawling out ratio of group A cells is less than 20%; use the corresponding culture medium of step 6 to replace the medium; Step 9: 72 hours after step 8, observe the crawling out of group A cells under a microscope. If the crawling out ratio of group A cells is greater than 75%, it is determined that the cells can be passaged. Step 10: Subculture the cells of group A; tap the culture bottle lightly to remove the tissue, add 6 ml of saline and shake lightly, pour out the saline and tissue, add 2 ml of GIAICO digestion solution, observe under the microscope, after the cells become round and suspended, add 10 ml of physiological saline to dilute, and then aspirate the suspension into a 50 ml centrifuge tube; adjust the balance according to the number of tubes to collect the suspension, centrifuge at 1500 rpm for 5 minutes; Step 11: After centrifugation, count the total number of cells in group A; Step 12: Take 2 million cells from group A, suspend them in 50 ml of complete culture medium, and plant them in a 175 cm 2 Place 25 ml of complete culture medium in a cell culture bottle and culture it in a 37°C, 5% CO2 incubator; this is the P1 generation of cells; Step 13: After 48 hours, subculture the cells in group A; pour out the complete medium, add 6 ml of saline and shake gently to pour out evenly, add 3 ml of GIAICO digestion solution, evenly spread the bottom of the culture bottle, observe under the microscope, wait for the cells to become round and fall off, add 15 ml of physiological saline to dilute, and aspirate the suspension into a 50 ml centrifuge tube; adjust the balance according to the number of tubes to collect the suspension, and centrifuge for 5 minutes at 1500 rpm; Step 14: Count and take out 3 million cells from group A, suspend them in 75 ml of complete medium, and plant them in three 175 cm 2 The cell culture flask was placed in a 37°C, 5% CO2 incubator for culture; this is the P2 generation of cells; Step 15: By repeating the operations of step 13 and step 14, P3 generation cells, P4 generation cells, and P5 generation cells are obtained in sequence; Step 16: After 48 hours, take a 4X photo of the cell morphology of group A; repeat step 13, collect 3 million cells from group A for flow cytometry identification, and take another 3 million cells for osteogenic, adipogenic and chondrogenic differentiation induction experiments; take another 3 million cells for soft agar clone microscopy tumorigenicity identification experiments.

2. The method for rapidly preparing umbilical cord-derived mesenchymal stem cells according to claim 1, characterized in that: The experimental reagents and consumables are as follows: HeliosUItraGRO serum replacement; GIBICO α-MEM medium; 10cm culture dish; Corning 75 and 175cm 2 Cell culture flask; sterile surgical scissors; tweezers; Corning 50ml centrifuge tube; 0.9% saline; 75% medical alcohol; GIBICO digestion solution; recombinant human endothelial cell inhibitory factor; THERMO centrifuge; THERMO biological safety cabinet; Pasteur pipette; 1ml and 10ml pipettes.

3. The method for rapidly preparing umbilical cord-derived mesenchymal stem cells according to claim 1, characterized in that: In the step 15, the acquisition interval of the P3 generation cells, the P4 generation cells, and the P5 generation cells is 48 hours.

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