Efficient production process of umbilical cord mesenchymal stem cell preparation

Abstract

The invention discloses an efficient production process of an umbilical cord mesenchymal stem cell preparation in the field of stem cells, which comprises the following steps: S101, collection and separation of umbilical cord mesenchymal stem cells: under a sterile condition, separating Walton's jelly by adopting a tissue block culture method, taking out an umbilical cord from an umbilical cord bottle, transferring the umbilical cord into a sterile culture dish, and reserving a sample of a protective solution in the umbilical cord bottle for sterile detection; washing the umbilical cord with normal saline, cutting off two ends of the umbilical cord, discarding the umbilical cord, segmenting the umbilical cord for about 3-4cm, washing to remove blood, umbilical cord veins, two arteries and amniotic membranes, collecting the Walton's jelly, and transferring the Walton's jelly into a 50ml centrifugal tube; adding a small amount of the culture medium, cutting the Walton's gum into pieces with a size of about 1mm < 3 > by using scissors, and centrifugally washing for two times by using normal saline; the preparation produced by the invention is negative in detection of hepatitis B virus, hepatitis C virus, human immunodeficiency virus, syphilis antibody, cytomegalovirus and mycoplasma, and is high in extraction efficiency and convenient to popularize.

Description

Technical Field

[0001] The present invention relates to the field of stem cells, and in particular to a high-efficiency production process of umbilical cord mesenchymal stem cell preparations. Background Art

[0002] Umbilical cord mesenchymal stem cells are primitive cells with the potential for self-replication and differentiation. They are special biological products with activity. The inconsistent cell sources and characteristics and the lack of unified standards for production and preparation methods will lead to fluctuations and differences in their therapeutic effects. It can be seen that the lack of standards makes it difficult for stem cell biomedical technology to be approved for clinical transformation; since there is no approval and no basis, the production process of stem cell preparations cannot be approved, which undoubtedly restricts the development of stem cell clinical transformation. Therefore, it is urgent to formulate a unified industry standard and then develop safe and quality-controlled stem cell preparations for clinical applications.

[0003] Therefore, those skilled in the art provide an efficient production process for umbilical cord mesenchymal stem cell preparations to solve the problems raised in the above background technology. Summary of the invention

[0004] The purpose of the present invention is to provide an efficient production process for umbilical cord mesenchymal stem cell preparations to solve the problems raised in the above background technology.

[0005] To achieve the above object, the present invention provides the following technical solutions:

[0006] The efficient production process of umbilical cord mesenchymal stem cell preparations includes the following steps:

[0007] S101. Collection and separation of umbilical cord mesenchymal stem cells:

[0008] Under sterile conditions, the tissue block culture method is used to separate Walton's jelly. The umbilical cord is removed from the umbilical cord bottle and transferred to a sterile culture dish. The protective fluid in the umbilical cord bottle is kept for sterility testing. The umbilical cord is washed with normal saline, and the two ends of the umbilical cord are cut off and discarded. The length of the segment is about 3-4cm, and the blood is washed away. The umbilical cord vein, 2 arteries, and amnion are removed. The Walton's jelly is collected and transferred to a 50ml centrifuge tube. Add a little culture medium, cut the Walton's jelly into pieces with scissors, the size is about 1mm3, and the saline is centrifuged and washed twice. Add culture medium and mix well, transfer to a culture bottle, put it in an incubator, and perform primary culture and record it as P0 generation. Static culture, change the medium according to the culture medium every 4-5 days, and continue to culture. A small amount of stem cells will grow on the 8th to 9th day, and the medium will be changed. The P0 generation cells of the tissue block culture method grow unevenly, and the subculture time is selected according to the cell growth. The culture condition of umbilical cord mesenchymal stem cells is a serum-free mesenchymal stem cell-specific culture medium, which does not contain human or animal serum;

[0009] S102, subculture:

[0010] After about 15 days of primary culture, gently tap the culture bottle, keep a sample of the supernatant for sterility testing, transfer the supernatant containing tissue blocks to a 50ml tube, centrifuge and take out the tissue blocks for a second culture, record it as the second P0 generation. Wash the culture bottle once with DPBS, suck it clean with a pipette, add 0.25% trypsin, incubate in a 37°C incubator, observe the cells become round under a microscope, gently tap the culture bottle until all the cells fall off, terminate digestion, centrifuge and wash to remove the supernatant, repeat the washing once (depending on the number of tissue blocks, you can choose whether to filter appropriately), choose the number of inoculation bottles according to the number of cells (generally human umbilical cord mesenchymal stem cells are inoculated at a cell concentration of 1 to 2×104 / cm2), transfer and inoculate into a new culture bottle, put it in an incubator for culture, record it as the P1 generation, expand the culture, and according to the cell growth, the cell fusion degree reaches 80% in about 3 days, and then carry out subculture and expansion culture, record it as the P2 generation (the supernatant of the subculture is kept for sterility testing). Carry out cell subculture in this way;

[0011] S105, Identification of biological characteristics of umbilical cord mesenchymal stem cells;

[0012] S106, quality control testing of umbilical cord mesenchymal stem cells;

[0013] S107. Umbilical cord mesenchymal stem cell storage.

[0014] As a further solution of the present invention: the umbilical cord mesenchymal stem cells in step S101 are collected under sterile conditions, specifically comprising the following steps:

[0015] S1011. First, after the fetus is delivered, the umbilical cord is cut as usual. Within 10 seconds after the fetus is delivered, two hemostats are used to clamp the fetus 5-8 cm from the umbilical cord and the umbilical cord is cut in the middle.

[0016] S1012. Wipe the umbilical cord with sterile gauze, and wipe the cut end of the umbilical cord with gauze disinfected with iodine or alcohol;

[0017] S1013. Umbilical cord blood sampling: 5 ml of umbilical cord blood is collected from umbilical cord blood vessels for hospital microbiological testing, and the mother's name and collection time are marked;

[0018] S1014, tie a ligature above the hemostat and cut off the section with the blood collection needle hole; clamp the umbilical cord with two hemostat 2-3 cm near the placenta end, then tie it with a ligature silk thread near the hemostat, and cut the umbilical cord between the two hemostats with surgical scissors;

[0019] S1015. Cut off an umbilical cord longer than 15 cm without pinholes and place it vertically into an umbilical cord collection bottle, minimizing contact with the bottle mouth and the outside, and mark the mother's name, collection time and other key information on the umbilical cord collection bottle;

[0020] S1016. Finally, fill in the “Collection Registration Form”.

[0021] As a further solution of the present invention: in step S101, the collected umbilical cord sample needs to be tested and a report needs to be issued.

[0022] As a further solution of the present invention: in step S1015, if a normal delivery is required, the umbilical cord is soaked in 75% alcohol for 2 minutes and then rinsed with saline.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] The preparation produced by the invention is negative in the detection of hepatitis B virus, hepatitis C virus, HIV, syphilis antibody, cytomegalovirus and mycoplasma, and has high extraction efficiency, which is convenient for promotion. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 It is a schematic diagram of the umbilical cord collection method of the present invention;

[0026] Figure 2 This is the stem cell collection registration form of the present invention;

[0027] Figure 3 This is a flow cytometry identification diagram of Huc-MSCsP3 in the present invention;

[0028] Figure 4 This is a diagram for identifying the adipogenic, osteogenic and chondrogenic differentiation of Huc-MSCs in the present invention. DETAILED DESCRIPTION

[0029] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0030] See also Figures 1 to 4 In an embodiment of the present invention, the efficient production process of umbilical cord mesenchymal stem cell preparation comprises the following steps:

[0031] S101. Collection and separation of umbilical cord mesenchymal stem cells:

[0032] Under sterile conditions, the tissue block culture method is used to separate Walton's jelly. The umbilical cord is removed from the umbilical cord bottle and transferred to a sterile culture dish. The protective fluid in the umbilical cord bottle is kept for sterility testing. The umbilical cord is washed with normal saline, and the two ends of the umbilical cord are cut off and discarded. The length of the segment is about 3-4cm, and the blood is washed away. The umbilical cord vein, 2 arteries, and amnion are removed. The Walton's jelly is collected and transferred to a 50ml centrifuge tube. Add a little culture medium, cut the Walton's jelly into pieces with scissors, the size is about 1mm3, and the saline is centrifuged and washed twice. Add culture medium and mix well, transfer to a culture bottle, put it in an incubator, and perform primary culture and record it as P0 generation. Static culture, change the medium according to the culture medium every 4-5 days, and continue to culture. A small amount of stem cells will grow on the 8th to 9th day, and the medium will be changed. The P0 generation cells of the tissue block culture method grow unevenly, and the subculture time is selected according to the cell growth. The culture condition of umbilical cord mesenchymal stem cells is a serum-free mesenchymal stem cell-specific culture medium, which does not contain human or animal serum;

[0033] S102, subculture:

[0034] After about 15 days of primary culture, gently tap the culture bottle, keep a sample of the supernatant for sterility testing, transfer the supernatant containing tissue blocks to a 50ml tube, centrifuge and take out the tissue blocks for a second culture, record it as the second P0 generation. Wash the culture bottle once with DPBS, suck it clean with a pipette, add 0.25% trypsin, incubate in a 37°C incubator, observe the cells become round under a microscope, gently tap the culture bottle until all the cells fall off, terminate digestion, centrifuge and wash to remove the supernatant, repeat the washing once (depending on the number of tissue blocks, you can choose whether to filter appropriately), choose the number of inoculation bottles according to the number of cells (generally human umbilical cord mesenchymal stem cells are inoculated at a cell concentration of 1 to 2×104 / cm2), transfer and inoculate into a new culture bottle, put it in an incubator for culture, record it as the P1 generation, expand the culture, and according to the cell growth, the cell fusion degree reaches 80% in about 3 days, and then carry out subculture and expansion culture, record it as the P2 generation (the supernatant of the subculture is kept for sterility testing). Carry out cell subculture in this way;

[0035] The umbilical cord mesenchymal stem cells in step S101 are collected under sterile conditions, and specifically include the following steps:

[0036] S1011. First, after the fetus is delivered, the umbilical cord is cut as usual. Within 10 seconds after the fetus is delivered, two hemostats are used to clamp the fetus 5-8 cm from the umbilical cord and the umbilical cord is cut in the middle.

[0037] S1012. Wipe the umbilical cord with sterile gauze, and wipe the cut end of the umbilical cord with gauze disinfected with iodine or alcohol;

[0038] S1013. Umbilical cord blood sampling: 5 ml of umbilical cord blood is collected from umbilical cord blood vessels for hospital microbiological testing, and the mother's name and collection time are marked;

[0039] S1014, tie a ligature above the hemostat and cut off the section with the blood collection needle hole; clamp the umbilical cord with two hemostat 2-3 cm near the placenta end, then tie it with a ligature silk thread near the hemostat, and cut the umbilical cord between the two hemostats with surgical scissors;

[0040] S1015. Cut off an umbilical cord longer than 15 cm without pinholes and place it vertically into an umbilical cord collection bottle, minimizing contact with the bottle mouth and the outside, and mark the mother's name, collection time and other key information on the umbilical cord collection bottle;

[0041] S1016. Finally, fill in the “Collection Registration Form”.

[0042] In the step S101, the collected umbilical cord sample needs to be tested and a report needs to be issued.

[0043] Wherein, in step S1015, if the delivery is normal, the umbilical cord needs to be immersed in 75% alcohol for 2 minutes and then rinsed with saline.

[0044] In this example, the biological characteristics of the collected umbilical cord mesenchymal stem cells were identified as follows:

[0045] Huc-MSCs phenotype identification:

[0046] The cell surface markers of three batches of third-generation Huc-MSCs were detected by flow cytometry (FCM), and the results showed that Huc-MSCs expressed high levels of CD73, CD90, and CD105, but were negative for CD34, CD45, and human leukocyte antigen (HLA)-DR, such as Figure 3 As shown, it can be seen that mesenchymal stem cells, rather than hematopoietic stem cells, were isolated and cultured in Wharton's jelly of human umbilical cord tissue.

[0047] Identification of Huc-MSCs differentiation into adipogenesis, osteogenic and chondrogenic

[0048] In order to determine whether Huc-MSCs have multidirectional differentiation potential, they were induced to differentiate into adipogenic, osteogenic, and chondrogenic pathways. Figure 4 As shown in Figure 2, after about 21 days of adipogenic induction, Huc-MSCs were observed to contain large and small fat vesicles in the cells under a microscope, and could be stained with Oil Red O staining solution ( Figure 4 A), confirming that the Huc-MSCs we cultured have the ability to differentiate into adipocytes; after about 28 days of adipogenic induction, a large number of bone nodules stained with alizarin red were observed in the cells under the microscope ( Figure 4B), confirming that the Huc-MSCs we cultured have the ability to differentiate into osteoblasts; after about 28 days of chondrogenic induction, the cells were observed to contain a large amount of cartilage acidic proteoglycans stained with alizarin red under the microscope, confirming that the Huc-MSCs we cultured have the ability to differentiate into chondrogenesis ( Figure 4 C), no cell differentiation was observed in the negative control group ( Figure 4 D).

[0049] In this embodiment, the quality control of the collected umbilical cord mesenchymal stem cells was tested:

[0050] Bacterial detection: Huc-MSCs cell culture supernatant was cultured in thioglycollate fluid medium, tryptic soy liquid medium and Sabouraud dextrose agar medium for 3, 7 and 14 days, and no bacteria grew, and the medium did not become turbid. As shown in the following table:

[0051]

[0052]

[0053] Endotoxin detection: Huc-MSCs cell culture supernatant was detected by horseshoe crab reagent gel method (horse crab reagent sensitivity λ = 0.025EU / ml). After completing the test of horseshoe crab reagent according to the kit operating requirements, the test tube was gently taken out and slowly inverted 180°. If gel was formed in the tube and the gel did not deform or slide off the tube wall, it was positive. If no gel was formed or the formed gel was not solid, deformed and slipped off the tube wall, it was negative. In this study, no gel was formed in the 6 cell supernatant samples from 3 sample sources, and the endotoxin test results were all negative, as shown in the following table:

[0054] Detection Reagents Limulus test kit Days 3 days 7 days N69020220000200 sample a (—)(—) N69020220000200 sample b (—)(—) N69020220001200 sample a (—)(—) N69020220001200 sample b (—)(—) N69020220001300 Sample a (—)(—) N69020220001300 sample b2 (—)(—)

[0055] Virus and mycoplasma detection of Huc-MSCs culture supernatant:

[0056] The quality control team of the NewSino laboratory tested the culture supernatants of the three groups of Huc-MSCs for hepatitis B virus, hepatitis C virus, HIV, syphilis antibodies, cytomegalovirus, and mycoplasma, and all were negative.

[0057] 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. An efficient production process for umbilical cord mesenchymal stem cell preparations, characterized by: The steps include: S101. Collection and separation of umbilical cord mesenchymal stem cells: Under sterile conditions, the Walton's jelly is separated by tissue block culture method, the umbilical cord is taken out from the umbilical cord bottle and transferred to a sterile culture dish, the protective fluid in the umbilical cord bottle is kept for sterility testing, the umbilical cord is washed with physiological saline, the two ends of the umbilical cord are cut off and discarded, the length of the segment is about 3-4 cm, the blood is washed, the umbilical cord vein, 2 arteries, and amniotic membrane are removed, the Walton's jelly is collected and transferred to a 50 ml centrifuge tube, a small amount of culture medium is added, the Walton's jelly is cut into pieces with scissors, the size is about 1 mm3, the physiological saline is centrifuged and washed twice, the culture medium is added and mixed, and the jelly is transferred to a culture bottle, which is placed in an incubator for primary culture and recorded as P0 generation, static culture, and the medium is changed according to the medium condition for 4-5 days. The culture is continued. A small amount of stem cells will grow on the 8-9th day, and the medium is changed. The P0 generation cells of the tissue block culture method grow unevenly, and the subculture time is selected according to the cell growth condition. The culture condition of umbilical cord mesenchymal stem cells is a serum-free mesenchymal stem cell-specific culture medium, which does not contain human or animal serum; S102, subculture: After about 15 days of primary culture, gently tap the culture bottle, keep the supernatant sample for sterility test, transfer the supernatant containing tissue blocks to a 50ml tube, centrifuge and take the tissue blocks for 2nd culture, record the second P0 generation, wash the culture bottle once with DPBS, suck it clean with a pipette, add 0.25% trypsin, incubate in a 37℃ incubator, observe the cells become round under a microscope, gently tap the culture bottle, all the cells fall off, terminate digestion, centrifuge and wash to remove the supernatant, repeat washing once, select the number of inoculation bottles according to the number of cells, transfer and inoculate into a new culture bottle, put it in the incubator for culture and record it as P1 generation, expand the culture, according to the cell growth, about 3 days later, the cell fusion degree reaches 80%, and then subculture and expand the culture, record it as P2 generation, and so on for cell subculture; S105, Identification of biological characteristics of umbilical cord mesenchymal stem cells; S106, quality control testing of umbilical cord mesenchymal stem cells; S107. Umbilical cord mesenchymal stem cell storage.

2. The efficient production process of umbilical cord mesenchymal stem cell preparation according to claim 1, characterized in that: The umbilical cord mesenchymal stem cells in step S101 are collected under sterile conditions and specifically include the following steps: S1011. First, after the fetus is delivered, the umbilical cord is cut as usual. Within 10 seconds after the fetus is delivered, two hemostats are used to clamp the fetus 5-8 cm from the umbilical cord and the umbilical cord is cut in the middle. S1012. Wipe the umbilical cord with sterile gauze, and wipe the cut end of the umbilical cord with gauze disinfected with iodine or alcohol; S1013. Umbilical cord blood sampling: 5 ml of umbilical cord blood is collected from umbilical cord blood vessels for hospital microbiological testing, and the mother's name and collection time are marked; S1014, tie a ligature above the hemostat and cut off the section with the blood collection needle hole; clamp the umbilical cord with two hemostat 2-3 cm near the placenta end, then tie it with a ligature silk thread near the hemostat, and cut the umbilical cord between the two hemostats with surgical scissors; S1015. Cut off an umbilical cord longer than 15 cm without pinholes and place it vertically into an umbilical cord collection bottle, minimizing contact with the bottle mouth and the outside, and mark the mother's name, collection time and other key information on the umbilical cord collection bottle; S1016. Finally, fill in the “Collection Registration Form”.

3. The efficient production process of umbilical cord mesenchymal stem cell preparation according to claim 1, characterized in that: In step S101, the collected umbilical cord sample also needs to be tested and a report issued.

4. The efficient production process of umbilical cord mesenchymal stem cell preparation according to claim 2, characterized in that: In step S1015, if the delivery is normal, the umbilical cord needs to be soaked in 75% alcohol for 2 minutes and then rinsed with saline.

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