CN115518076A - New generation stem cell therapeutic agent for treating gonarthritis - Google Patents
New generation stem cell therapeutic agent for treating gonarthritis Download PDFInfo
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Abstract
The application discloses a new generation stem cell therapeutic agent for treating gonitis, which relates to the stem cell field and comprises the following components: preparing collagenase: dissolving 100mg collagenase I powder in 100ml PBS containing calcium and magnesium, filtering with a filter, subpackaging, and keeping in the dark for later use; preparing a stem cell complete culture medium: serum-free medium + serum replacement; preparing pancreatin-EDTA: diluting pancreatin-EDTA with DPBS; in the invention, the human umbilical cord mesenchymal stem cells used have the characteristics of strong proliferation and differentiation capacity, low immunogenicity and difficult occurrence of graft-versus-host disease after transplantation; the optimal condition for inducing umbilical cord mesenchymal stem cells to differentiate towards cartilage is determined by analyzing the co-action of intercellular connection, protein and the like.
Description
Technical Field
The invention belongs to the technical field of stem cells, and particularly relates to a new generation stem cell therapeutic agent for treating gonitis.
Background
Most of the cell therapeutic agents currently on the market are stem cell studies and preparations using patient's own extracted tissues. This additional surgery causes additional physical trauma to the patient. If the cartilage is regenerated, the complex tissue structure of the cartilage must be reconstructed, and the process is complicated and long in time. At present, a biological tissue engineering scaffold highly suitable for human cartilage is not found, the cartilage is not only composed of cells, but also contains various substances such as collagen fibers, proteoglycan and the like, and stem cells cannot stay in joint cavities for too long time, so that the repair effect is slight.
Disclosure of Invention
The technical problem to be solved is as follows:
aiming at the defects of the prior art, the invention provides a new generation of stem cell therapeutic agent for treating gonitis, and solves the problems mentioned in the background technology.
The technical scheme is as follows:
in order to realize the purpose, the invention is realized by the following technical scheme:
a new generation of stem cell therapeutic agent for treating gonarthritis comprises the following steps:
A. preparation of the solution
A1, preparation of collagenase: dissolving 100mg collagenase I powder in 100ml PBS containing calcium and magnesium, filtering with a filter, subpackaging, and keeping in the dark for later use;
a2, preparing a stem cell complete culture medium: serum-free medium + serum replacement;
a3, preparing pancreatin-EDTA: diluting pancreatin-EDTA with DPBS;
B. preparation of umbilical cord MSCs
B1, taking umbilical cord tissues of healthy lying-in women after 39-40 weeks of normal delivery or caesarean delivery;
b2, filling the umbilical cord tissue with umbilical cord preservation solution, sealing the umbilical cord tissue in a preservation bottle, and then transferring the umbilical cord tissue to a laboratory;
b3, cleaning blood stain on the surface of the umbilical cord, cutting the umbilical cord into small sections by using surgical scissors, cutting the umbilical cord by using sterile scissors along the parallel direction of venous blood vessels, tearing off a venous membrane along the cut edge by using a hooked tooth forceps, then flatly spreading the umbilical cord, and searching for two arterial blood vessels; removing two arteries in the umbilical cord by using hook tooth forceps, wherein the rest part is Wharton's jelly and the amniotic membrane on the surface of the umbilical cord; separating the Wharton's jelly by using two hook tooth tweezers, and stripping the jelly;
b4, placing the separated Wharton's jelly in a sterilized beaker, and shearing the Wharton's jelly into tissue blocks as small as possible by using surgical scissors;
b5, transferring the tissue block into a centrifuge tube, adding a corresponding amount of collagenase I, placing the tissue block into a 37-DEG carbon dioxide incubator for enzymolysis, transferring the tissue block into a cell sieve after enzymolysis, filtering the tissue block into a new centrifuge tube, supplementing physiological saline for centrifugation, sucking and removing supernatant, adding growth medium to suspend cells, transferring the cells into a culture dish after resuspension, and placing a culture bottle into the carbon dioxide incubator for culture;
b6, the culture bottle is not required to be shaken, the single cells are fully attached to the wall, the liquid is changed subsequently, the fresh MSC culture medium is changed, and the culture is placed in a carbon dioxide incubator for culture;
b7, observing the growth condition of the MSCs cell colony in the culture dish, and executing P0-P1 passage operation according to the growth condition of the cell colony; digestion with pancreatin solution: removing the cell culture solution in the culture dish by using a pipettor, adding physiological saline into the culture dish, slightly shaking the culture dish, discarding the physiological saline, and repeatedly cleaning once; adding pancreatin liquid into the culture dish, standing at 37 ℃, and observing the cell digestion condition under a microscope; when most of cells are detached from the wall, collecting cell suspension into a centrifuge tube, collecting the cells, and removing supernatant; washing the collected cells once with normal saline, and discarding the supernatant; adding a pre-warmed subculture medium into a centrifuge tube filled with cells, resuspending the cells, inoculating the cells into two culture dishes, adding the pre-warmed subculture medium into each culture dish, and culturing in a carbon dioxide incubator;
b8, observing the growth condition of the MSCs every day, performing passage amplification when the cell density reaches a specified concentration, and performing P1-P2 passage amplification;
and B9, adding the chondrocyte induction reagent into the umbilical cord mesenchymal stem cells for induction culture.
In one possible implementation, in B1, the parturient needs to detect HIV virus, TP virus, HAV virus, HBV virus, HCV virus, all five of which are negative and have no history of infection.
In a possible implementation, in B2, the operating room needs to be kept clean, and the storage bottles need to be transported to the laboratory at 2-8 ℃.
In a possible implementation mode, in B3, before the blood stain on the surface of the umbilical cord is cleared, the umbilical cord is placed in a super clean bench, after being soaked and disinfected by iodine, the umbilical cord is taken out by using sterile beard teeth forceps, is placed in a culture dish containing physiological saline prepared in advance, is cleaned for 2-3 times, and is placed in 5 dishes of 10cm at most, and the length of the umbilical cord is measured.
In a possible implementation, in B3, the gel is stripped off with care being taken not to be associated with the amniotic membrane layer.
In a possible implementation manner, in the step B5, in the enzymolysis process, the enzymolysis solution is repeatedly blown by a pipette while intermittently and violently shaking once to observe the enzymolysis effect.
Has the beneficial effects that:
in the invention, the human umbilical cord mesenchymal stem cells used have the characteristics of strong proliferation and differentiation capacity, low immunogenicity and difficult occurrence of graft-versus-host disease after transplantation;
determining the optimal condition for inducing umbilical cord mesenchymal stem cells to differentiate into cartilage by analyzing the co-action of intercellular connection, protein and the like;
the cell layer plate technology is utilized to reconstruct the cartilage tissue structure, and the in-vivo inflammation regulation and control environment is simulated through the cell co-culture technology, so that the preparation scheme of the tissue maturity predictability regeneration cartilage transplantation preparation is formulated, the cartilage growth environment is stably repaired, and the repair curative effect is evaluated.
Drawings
FIG. 1 is a human primary umbilical cord mesenchymal stem cell (X40) in the first embodiment of the present invention;
FIG. 2 shows colony formation (X100) after passaging cells in the first example of the present invention;
FIG. 3 shows that human umbilical cord mesenchymal stem cells are fused by more than 90% in the first embodiment of the present invention;
FIG. 4 shows cartilage visible after induction culture of cells according to one embodiment of the present invention;
FIG. 5 is a comparison graph of HE staining of a section of a tissue block according to a second embodiment of the present invention;
FIG. 6 is a graph showing HE staining of a slice of an induced osteoblast layer plate in the second embodiment of the present invention;
FIG. 7 is a graph of the staining of the induced cartilage A Li Xinlan in example two of the present invention;
FIG. 8 is a graph of a Li Xinlan control staining according to example two of the present invention;
FIG. 9 is a graph of staining of the osteoblast-induced layer Li Xinlan in example two of the present invention;
FIG. 10 is a graph of alizarin red staining by bone formation induction in the second embodiment of the present invention;
FIG. 11 is a comparison chart of the staining of alizarin red induced by osteogenesis in the second embodiment of the present invention;
FIG. 12 is a graph showing Safranin-O staining induced by osteogenesis in example two of the present invention;
FIG. 13 is a comparison graph of Safranin-O staining of osteogenic induction in example two of the present invention;
FIG. 14 is a Safranin-O staining pattern of the osteoblast layer induced in the second example of the present invention;
FIG. 15 is a comparison of Mr. plum before and after treatment in a third patient according to the present invention;
FIG. 16 is a comparison of Mr. wang before and after treatment in example III of the present invention;
FIG. 17 is a comparison of treatment before and after treatment of Mr. Lu in a third patient according to this embodiment of the present invention;
FIG. 18 is a data Table showing the real-time quantitative PCR primer sequences Table 2 PCR primers and sequences for SOX9, col1a1 and Col2a1, col1a1 and dCol2a1 in example three of the present invention;
FIG. 19 is a histogram of the expression levels of SOX9 and Col1a1 in example three of the present invention;
FIG. 20 is a histogram of a neutralization Col2a1 according to example three of the present invention;
FIG. 21 is a graph showing the expression levels of SOX9, type I collagen and type II collagen in the third example of the present invention;
FIG. 22 is a photograph showing the detection of a cell surface antigen marker in the third embodiment of the present invention;
FIG. 23 is a photograph showing immunofluorescence assay (X100) of chondrocytes in the third example of the present invention.
Detailed Description
The embodiments of the present application solve the problems of the prior art by providing a new generation of stem cell therapeutic agent for treating gonarthritis.
Example 1
As shown in fig. 1 and 4, a new generation of stem cell therapeutic agent for treating gonarthritis includes the following steps:
A. preparation of the solution
A1, preparation of collagenase: dissolving 100mg collagenase I powder in 100ml PBS containing calcium and magnesium, filtering with a filter, subpackaging, and keeping in the dark for later use;
a2, preparing a stem cell complete culture medium: serum-free medium + serum replacement;
a3, preparing pancreatin-EDTA: diluting pancreatin-EDTA with DPBS;
B. preparation of umbilical cord MSCs
B1, taking umbilical cord tissues of healthy lying-in women after 39-40 weeks of normal delivery or caesarean delivery;
b2, filling the umbilical cord tissue with umbilical cord preservation solution, sealing the umbilical cord tissue in a preservation bottle, and then transferring the umbilical cord tissue to a laboratory;
b3, cleaning blood stain on the surface of the umbilical cord, cutting the umbilical cord into small sections by using surgical scissors, cutting the umbilical cord by using sterile scissors along the parallel direction of venous blood vessels, tearing off a venous membrane along the cut edge by using a hooked tooth forceps, then flatly spreading the umbilical cord, and searching for two arterial blood vessels; removing two arteries in the umbilical cord by using hook tooth forceps, wherein the rest part is Wharton's jelly and the amniotic membrane on the surface of the umbilical cord; separating the Wharton's jelly by using two hook tooth tweezers, and stripping the jelly;
b4, placing the separated Wharton's jelly in a sterilized beaker, and shearing the Wharton's jelly into tissue blocks as small as possible by using surgical scissors;
b5, transferring the tissue block into a centrifuge tube, adding a corresponding amount of collagenase I, placing the tissue block into a 37-DEG carbon dioxide incubator for enzymolysis, transferring the tissue block into a cell sieve after enzymolysis, filtering the tissue block into a new centrifuge tube, supplementing physiological saline for centrifugation, sucking and removing supernatant, adding growth medium to suspend cells, transferring the cells into a culture dish after resuspension, and placing a culture bottle into the carbon dioxide incubator for culture;
b6, a culture bottle is not required to be shaken to ensure that the single cells are fully attached to the wall, the liquid is changed subsequently, a fresh MSC culture medium is changed, and the culture medium is placed in a carbon dioxide incubator for culture;
b7, observing the growth condition of the MSCs cell colony in the culture dish, and executing P0-P1 passage operation according to the growth condition of the cell colony; digestion with pancreatin liquid: removing the cell culture solution in the culture dish by using a pipettor, adding physiological saline into the culture dish, slightly shaking the culture dish, removing the physiological saline, and repeatedly cleaning once; adding pancreatin liquid into the culture dish, standing at 37 ℃, and observing the cell digestion condition under a microscope; when most of cells are detached from the wall, collecting cell suspension into a centrifuge tube, collecting the cells, and removing supernatant; washing the collected cells once with normal saline, and discarding the supernatant; adding a pre-warmed subculture medium into a centrifuge tube filled with cells, resuspending the cells, inoculating the cells into two culture dishes, adding the pre-warmed subculture medium into each culture dish, and culturing in a carbon dioxide incubator;
b8, observing the growth condition of the MSCs every day, performing passage amplification when the cell density reaches a specified concentration, and performing P1-P2 passage amplification;
and B9, adding the chondrocyte induction reagent into the umbilical cord mesenchymal stem cells for induction culture.
In some examples, B1, the parturient needs to detect HIV, TP, HAV, HBV, HCV, all five of which are negative with no history of infection.
In some examples, in B2, the operating room needs to be kept clean and the storage bottles need to be transported to the laboratory at 2-8 ℃.
In some examples, in B3, before the blood stain on the surface of the umbilical cord is cleared, the umbilical cord is placed in a clean bench, after soaking and sterilization with iodine, the umbilical cord is taken out with a sterile pair of forceps, placed in a culture dish containing physiological saline prepared in advance, washed 2 to 3 times, at most 5 dishes of 10cm, and the length of the umbilical cord is measured.
In some cases, in B3, care was taken not to accompany the amniotic membrane layer when peeling the gel.
In some examples, B5, during the enzymolysis, shaking vigorously once at intervals, observing the enzymolysis effect, and repeatedly blowing the enzymolysis solution with a pipette.
Example 2
Preparing a stem cell laminate for transplantation:
step one, using a temperature-mediated method for separating cells from a poly (n-isopropylacrylamide) (PIPAAM) grafting temperature response culture dish to automatically contract various intercellular surface layers and generate various intercellular effects;
step two, putting the cells harvested in the step one into a six-hole plate culture dish, and culturing in a CO2 and 37 ℃ culture apparatus by using an MSC cell culture solution;
step three, placing the cells harvested on the Pellet culture into a centrifugal tube for centrifugal separation, moving the cells to a culture dish, and culturing and inducing the cells to form cell particles;
step four, placing the cells subjected to monolayer culture into a cell culture dish at a certain concentration for culture;
step five, culturing the Cell sheet cells in a culture dish according to a certain concentration, separating a Cell layer, attaching the Cell layer to the Cell culture dish, culturing the Cell layer by using a chondrocyte differentiation culture solution, and creating 3-5 Cell layers according to requirements for use.
Referring to fig. 5-6, histological analysis: fixing the sample, washing, slicing, H-E staining, observing whether the basic structure and cartilage differentiation are carried out or not, and observing whether the main component glycoprotein of the cartilage matrix is stained and the degree by adopting a Safranin-O staining method;
conclusion from immunohistological analysis:
referring to fig. 7-9, the results of the staining with a Li Xinlan show that the staining of the experimental group is strong positive after induction, and the extracellular matrix is stained blue, indicating that it secretes a large amount of GAG; the control group was lightly colored (FIG. 1).
Referring to fig. 10-11, alizarin red staining results show that the staining of the experimental groups after induction is strong positive, and cells are stained orange red, which indicates that mineralized nodules are formed; the control group was very lightly colored (FIG. 2).
Referring to fig. 12-14, the results of oil red O staining show that the experimental groups were positive after induction, and bright red particles appeared in the cytoplasm of the cells, indicating that the cytoplasmic contents were fat droplets; the control group showed no coloration (FIG. 3)
Real-time quantitative PCR analysis
The cells in culture were treated with an RNA extraction kit to extract total RNA, and after synthesizing their cDNA by reverse transcription, quantitative polymerase chain reaction (qPCR) was performed for each cDNA.
The relative gene expression pattern is based on GAPDH expression.
Example 3
Stem cell characterization
Transcriptome analysis
Refer to FIG. 18
Real-time quantitative PCR detection of SOX9, COL1A1 and COL2A1mRNA expression, and extracting total cell RNA from chondrocytes before and after culture and after induced differentiation of human umbilical cord mesenchymal stem cells by using a Trizol kit according to instructions.
And (3) measuring the purity and the content of the RNA by a micro ultraviolet spectrophotometer (Nanodrop), wherein each RNA sample (A260/A280) is 1.7-2.1.
2.5 μ g RNA was taken and RNA was transcribed into cDNA according to the reverse transcription kit instructions. Real-time quantitative PCR detection is carried out by the Real-time timePCR kit.
Taking 1 microliter of the inverted cDNA product as a template, taking GAPDH as an internal reference, and detecting the relative expression amounts of SOX-9, type II collagen and type I collagen genes in different treatment groups by using SYBRGreen I dye real-time quantitative PCR.
The qRT-PCR reaction conditions were: pre-denaturation at 95 ℃ for 30s;95 ℃ for 5s,60 ℃ for 30s,40 cycles; 15s at 90 ℃;60 ℃ for 1min. The experiment was repeated 3 times, and the mean value was taken and GADPH was used as the internal reference. Data analysis was performed by the Δ Δ Ct method, and the data were averaged over 3 replicates using the primer sequences shown in the table.
Reference is made to FIGS. 19-20
Expression level of SOX9, col1a1 and Col2a1 mRNAs
Drawing notes: 1: human articular chondrocyte blank control; 2: human umbilical cord mesenchymal stem cell blank control; 3: a positive control of human umbilical cord mesenchymal stem cells; 4,5,6: the reagent dosage for inducing and culturing the human umbilical cord mesenchymal stem cells is 1: 1,3: 1,5: 1 respectively. The expression level of SOX9 mRNA in the induction group is higher than that in the positive control group (P < 0.05), and the relative expression level in the 3:1 induction group is the highest (P < 0.05); the expression quantity of Col1a 1mRNA in the 3:1 induction group is higher than that in the positive control group (P is less than 0.05); the proportion of human umbilical cord mesenchymal stem cells in the culture group is increased, and the expression quantity of Col2a1mRNA is reduced (P < 0.05). aP <0.05 compared to human articular chondrocyte blank; bP <0.05 compared to the positive control group;
protein analysis
And detecting the expression levels of SOX9, type I collagen and type II collagen by using Western-blot.
After induction culture, total cellular protein was extracted using RIPA lysine buffer.
And transferring the protein to a PVDF membrane after SDS-PAGE electrophoresis.
Exposure detection was performed with SOX9, COL1A1 and COL2A1 primary antibodies and HRP-bearing secondary antibody, respectively
Measuring the expression level of the protein.
Refer to FIG. 21
Detecting the expression quantity of SOX9, type I collagen and type II collagen;
note: 1: human articular chondrocyte blank control; 2: human umbilical cord mesenchymal stem cell blank control; 3: a positive control; 4: 1: 1 induction group; 5: 3:1 induction group; 6: 5: 1 induction group.
Cell viability phenotype assay
With reference to FIGS. 22-23, surface antigens (CD 105-PE, CD73-APC, CD34-PE, CD 45-FITC) were detected by flow cytometry.
Immunofluorescence detection of type II collagen, after induction culture, cells were washed 3 times with PBS, paraformaldehyde fixed, then washed 3 times with PBS, and treated with Triton X100 membrane-permeable.
Washed 3 more times with PBS and blocked with Bovine Serum Albumin (BSA) wet box at room temperature. PBS was washed 3 times and incubated overnight with type II collagen. PBS was washed 3 times and FITC-labeled secondary antibody was incubated at room temperature in the dark. PBS wash 3 times, DAPI incubate in the dark at room temperature, PBS wash 3 times, fluorescence microscopy and photograph.
In fig. 22, note: a is a target cell region; b is IgG1-PE and IgG1-FITC used for nonspecific binding control; c is CD34-PE and CD45-FITC; d is CD105-PE and CD73-FITC.
Note: a is human articular chondrocyte blank control; b is a human umbilical cord mesenchymal stem cell blank control; c is a positive control; d is an inducer group with the inducer dose of 1: 1; e is an induction group with the inducer dose of 3: 1; f is inducer dose 5: 1 induction group.
And (4) analyzing results:
the result of detecting cell surface antigen by using a flow cytometer as a human umbilical cord mesenchymal stem cell surface antigen marker shows that human umbilical cord mesenchymal stem cells highly express CD105 and CD73 and lowly express CD34 and CD45 (as shown in figure 22), meet the immunological characteristics of the mesenchymal stem cells, and induce 3: group 1 highly expressed the chondrocyte phenotype.
Qualitative detection result of type II collagen: the human umbilical cord mesenchymal stem cell blank control group shows negative immunofluorescence staining, the human articular chondrocyte blank control group, the human umbilical cord mesenchymal stem cell positive control group and the human articular chondrocyte induced group all show positive cell staining, cytoplasm and extracellular matrix all show red fluorescence (as shown in figure 23), the fluorescence signal of the induced group is strongest, and no obvious difference exists in the groups.
The SOX9 mRNA expression quantity of the real-time quantitative PCR detection SOX9, col1a1 and Col2a1mRNA expression induction culture groups is higher than that of the positive control group (P < 0.05), and the relative expression quantity of the 3:1 induction culture group is the highest (P < 0.05). In addition, the SOX9 mRNA expression level of the blank control group is lower than that of the positive control group (P < 0.05), and the expression level of the human umbilical cord mesenchymal stem cell blank control group is lowest (P < 0.05) (tables 20-21).
4.4Western-blot detection of SOX9, type I collagen and type II collagen expression: the expression level of the SOX9 protein in the induction group is higher than that of the positive control group, and no obvious difference exists in the group. The SOX9 protein expression level of the human articular chondrocyte blank control group is the highest, and the SOX9 protein expression level of the human umbilical cord mesenchymal stem cell blank control group is the lowest (as shown in figure 22).
Experimental data:
the following are some case data presentations:
patient manyflower willow: age 64;
symptoms are as follows: meniscus damage, pain;
treatment of the condition: the pain feeling is obviously relieved and the freedom of movement is improved after the stem cell treatment for 3 days.
The patient is old and born: 60 years old;
symptoms are: synovitis, fluid accumulation, pain;
the treatment conditions are as follows: after the stem cell treatment is carried out for 6 days, the effusion disappears, the pain is relieved, the subsequent state is good, and the movement is free.
Horse and woman patients: age 55;
symptoms are: traumatic arthritis, pain stiffness and swelling;
treatment of the condition: the pain, swelling and stiffness of the treated stem cells are reduced.
Korean women: 60 years old;
symptoms are as follows: synovitis, cartilage damage, fluid accumulation, pain;
treatment of the condition: the burning sensation is reduced, the effusion disappears and the pain is reduced after the stem cell treatment.
The patient Guo lady: age 67;
symptoms are: senile arthritis, pain, difficulty in starting;
treatment of the condition: the pain is reduced, the friction is reduced and the freedom of movement is improved after the stem cell therapy.
Patient mr. Plum: age 31 years old
The disease symptoms are as follows: medial meniscal tear
After stem cell treatment: one-year follow-up visit, pain disappeared, the activity was normal, and MRI showed that meniscus lesions healed.
The patient is a mankind: age 67 years old
The disease symptoms are as follows: knee osteoarthritis, pain, subchondral bone defect, cyst
After stem cell treatment: the pain is obviously improved after 13 months of follow-up, the cyst is obviously reduced, and the articular cartilage defect surface is reduced.
Lu Mir of the patient: age 48 years old
The disease symptoms are as follows: right ischemic femoral head necrosis with cyst, pain, restricted mobility for 6 months;
after stem cell treatment: the follow-up of 4 months, MRI examination cyst is reduced, and pain is obviously relieved.
Finally, it should be noted that: it should be understood that the above examples are only for clearly illustrating the present invention and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications of the invention may be made without departing from the scope of the invention.
Claims (6)
1. A new generation of stem cell therapeutic agent for treating gonitis, characterized by comprising the steps of:
A. preparation of the solution
A1, preparation of collagenase: dissolving 100mg collagenase I powder in 100ml PBS containing calcium and magnesium, filtering with a filter, subpackaging, and keeping in the dark for later use;
a2, preparing a stem cell complete culture medium: serum-free medium + serum replacement;
a3, preparing pancreatin-EDTA: diluting pancreatin-EDTA with DPBS;
B. preparation of umbilical cord MSCs
B1, taking umbilical cord tissues of healthy lying-in women after 39-40 weeks of normal delivery or caesarean delivery;
b2, filling the umbilical cord tissue with umbilical cord preservation solution, sealing the umbilical cord tissue in a preservation bottle, and then transferring the umbilical cord tissue to a laboratory;
b3, cleaning blood stain on the surface of the umbilical cord, cutting the umbilical cord into small sections by using surgical scissors, cutting the umbilical cord by using sterile scissors along the parallel direction of venous blood vessels, tearing off a venous membrane along the cut edge by using a hooked tooth forceps, then flatly spreading the umbilical cord, and searching for two arterial blood vessels; removing two arteries in the umbilical cord by using hook tooth forceps, wherein the rest part is Wharton's jelly and the amniotic membrane on the surface of the umbilical cord; separating the Wharton's jelly by using two hook tooth forceps, and stripping the jelly;
b4, placing the separated Wharton's jelly in a sterilized beaker, and shearing the Wharton's jelly into tissue blocks as small as possible by using surgical scissors;
b5, transferring the tissue block into a centrifuge tube, adding a corresponding amount of collagenase I, placing the tissue block into a 37-DEG carbon dioxide incubator for enzymolysis, transferring the tissue block into a cell sieve after enzymolysis, filtering the tissue block into a new centrifuge tube, supplementing physiological saline for centrifugation, sucking and removing supernatant, adding growth medium to suspend cells, transferring the cells into a culture dish after resuspension, and placing a culture bottle into the carbon dioxide incubator for culture;
b6, the culture bottle is not required to be shaken, the single cells are fully attached to the wall, the liquid is changed subsequently, the fresh MSC culture medium is changed, and the culture is placed in a carbon dioxide incubator for culture;
b7, observing the growth condition of the MSCs cell colony in the culture dish, and executing P0-P1 passage operation according to the growth condition of the cell colony; digestion with pancreatin solution: removing the cell culture solution in the culture dish by using a pipettor, adding physiological saline into the culture dish, slightly shaking the culture dish, discarding the physiological saline, and repeatedly cleaning once; adding pancreatin liquid into the culture dish, standing at 37 ℃, and observing the cell digestion condition under a microscope; when most of cells are detached from the wall, collecting cell suspension into a centrifuge tube, collecting the cells, and removing supernatant; washing the collected cells once with normal saline, and discarding the supernatant; adding a pre-warmed subculture medium into a centrifuge tube filled with cells, resuspending the cells, inoculating the cells into two culture dishes, adding the pre-warmed subculture medium into each culture dish, and culturing in a carbon dioxide incubator;
b8, observing the growth condition of the MSCs every day, performing passage amplification when the cell density reaches a specified concentration, and performing P1-P2 passage amplification;
and B9, adding the chondrocyte induction reagent into the umbilical cord mesenchymal stem cells for induction culture.
2. The new-generation stem cell therapeutic agent for the treatment of gonarthritis according to claim 1, wherein: in B1, the parturient needs to detect HIV virus, TP virus, HAV virus, HBV virus and HCV virus, all five viruses are negative, and no infection history exists.
3. The new-generation stem cell therapeutic agent for treating gonarthritis according to claim 1, characterized in that: in B2, the operating room needs to be kept clean, and the storage bottle needs to be transported to a laboratory at the temperature of 2-8 ℃.
4. The new-generation stem cell therapeutic agent for treating gonarthritis according to claim 1, characterized in that: and B3, before the blood stain on the surface of the umbilical cord is cleaned, placing the umbilical cord in a super clean bench, soaking and disinfecting the umbilical cord in iodine, taking out the umbilical cord by using sterile hook teeth to forceps, placing the umbilical cord in a culture dish containing physiological saline prepared in advance, cleaning the culture dish for 2 to 3 times, and measuring the length of the umbilical cord, wherein the maximum number of the dishes is 5 and 10 cm.
5. The new-generation stem cell therapeutic agent for the treatment of gonarthritis according to claim 1, wherein: in B3, the gel is stripped, and the amniotic membrane layer cannot be associated with the gel.
6. The new-generation stem cell therapeutic agent for treating gonarthritis according to claim 1, characterized in that: and B5, in the enzymolysis process, intermittently and violently shaking once, observing the enzymolysis effect, and repeatedly blowing and beating the enzymolysis liquid by using a pipette.
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Application publication date: 20221227 |