CN115216440A

CN115216440A - Method for improving umbilical cord mesenchymal stem cell yield in shorter culture time

Info

Publication number: CN115216440A
Application number: CN202110433951.8A
Authority: CN
Inventors: 王丽莎; 刘冬羽; 李治寰
Original assignee: Dongguan Enlian Stem Cell Biotechnology Research Institute
Current assignee: Dongguan Enlian Stem Cell Biotechnology Research Institute
Priority date: 2021-04-20
Filing date: 2021-04-20
Publication date: 2022-10-21

Abstract

The invention relates to a method for improving the yield of umbilical cord-derived mesenchymal stem cells in a short culture time. The main steps are that an umbilical cord tissue with the length of about 30cm is cut into 4-5 cm small sections, the blood is removed, the Fahrenheit jelly is stripped out after the blood is cleaned, the Fahrenheit jelly is put into a complete culture medium containing 10 percent blood substitute for soaking, and then the cutting is carried out until the size is 1-3 mm ³ The tissue blocks were then inoculated into culture flasks for culture. And harvesting the first primary umbilical cord mesenchymal stem cells after 8-12 days of culture, and simultaneously recovering tissue blocks for secondary adherent culture. And harvesting the second batch of primary umbilical cord mesenchymal stem cells after culturing for 5-9 days. By adopting the method, the cells in the Fahrenheit glue can be liberated to the maximum extent within 20 days, and the tissue utilization rate and the primary cell yield are improved; meanwhile, the operation steps are simple and convenient, and the cell collection operation can be completed only by two times.

Description

Method for improving umbilical cord mesenchymal stem cell yield in shorter culture time

Technical Field

The invention belongs to the technical field of biology, and relates to a method for improving the yield of umbilical cord mesenchymal stem cells in a short culture time.

Background

Mesenchymal Stem Cells (MSCs) are Cells with self-renewal and differentiation ability, can be differentiated into various tissue Cells such as fat, bone, cartilage, muscle, tendon, ligament, nerve, liver, cardiac muscle, endothelium and the like under specific induction conditions in vivo or in vitro, still have multidirectional differentiation potential after continuous subculture and cryopreservation, and can be used as ideal seed Cells for repairing tissue and organ injuries caused by aging and pathological changes. At present, mesenchymal stem cells have been widely used in clinical research, and have a wide application prospect in the treatment of various diseases such as arthritis, cerebral palsy, alzheimer's disease, cardiovascular diseases, diabetes, immune system diseases and the like. Mesenchymal stem cells have wide sources, and at present, the mesenchymal stem cells are successfully separated from tissues such as dental pulp, fat, bone marrow, umbilical cord, placenta and the like. The umbilical cord mesenchymal stem cells are derived from umbilical cord Fahrenheit glue, can be differentiated into various tissue cells, have the advantages of low immunogenicity, no need of matching during allograft, no damage to collectors, no ethical problem and the like, and are ideal materials for clinical cell therapy.

At present, methods for separating umbilical cord mesenchymal stem cells are mainly divided into two types, one type is an enzyme digestion culture method, and the other type is a tissue block adherent culture method. Because the cost of the enzyme digestion culture method is higher, collagenase has direct toxic effect on cells when digesting the collagen matrix, and the digestion time is difficult to control, thus easily causing low vitality and poor state of the harvested cells, and simultaneously easily introducing exogenous substances, and increasing the risk of clinical application; the tissue block wall-adhering method is simple, convenient and feasible in process and low in cost, so that the method is widely applied at present. However, the existing tissue mass adherence method for obtaining primary mesenchymal stem cells has some disadvantages, such as long time, small number of obtained cells, low tissue mass utilization rate, and the like, and in addition, from the perspective of clinical application, the larger the number of mesenchymal stem cells derived from the same umbilical cord, the more the mesenchymal stem cells can be used in clinic, and the more stable the quality of the new drug of stem cells produced, therefore, how to maximally separate the mesenchymal stem cells in the umbilical cord tissue in a short time becomes a problem to be solved urgently.

Disclosure of Invention

In order to solve the above problems, we invented a separation method that can improve the yield of primary umbilical cord mesenchymal stem cells in a shorter culture time. After the blood vessels of the umbilical cord tissue are removed and the amnion is torn off, the remaining tissue is Fahrenheit glue. Soaking Farfaran gum in complete culture medium at 20-37 deg.C for a period of time, removing complete culture medium, and performing surgical operationShearing Fahrenheit glue to 1-3 mm ³ And (4) organizing the blocks. Uniformly spreading the tissue mass at the bottom of the culture flask, and adding 5% of CO ₂ Culturing in an incubator at 37 ℃ for 0-24 hours, adding a complete culture medium for continuous culture, and replacing the complete culture medium once in 3-5 days. On the 10 th day of culture, the cell fusion degree can reach about 80%, and the tissue block and the cells are respectively collected. And (3) carrying out second adherent culture on the tissue block, wherein the cell fusion degree reaches about 80% after about 7 days of culture, and collecting the cells subjected to the second adherent culture. The cell morphology, the survival rate, the surface antigen expression condition and the trilineage differentiation capability of the cells collected twice have no obvious difference. By using the method, the cells in the Fahrenheit glue can be furthest dissociated within 20 days, and the tissue utilization rate and the primary cell yield are improved; meanwhile, the operation steps are simple and convenient, and the cell collection operation can be completed only by two times.

The complete culture medium adopted by the invention is a basic culture medium containing hemocyte, the hemocyte is preferably platelet lysate, the hemocyte content is 10%, and the basic culture medium is preferably alpha-MEM.

The soaking temperature of the Fahrenheit glue is preferably 25 ℃; the soaking time is 0 to 24 hours, preferably 20 to 30min.

The tissue block is cultured in the incubator for 8-16 hr and then added with complete culture medium to result in high culture effect.

The number of times of tissue block attachment is not limited to two in the present invention, but is preferably two; multiple attachments may be made until cells that have dissociated from the tissue mass are not harvested.

The invention has the advantages that: a large amount of primary mesenchymal stem cells can be harvested after the tissue blocks are subjected to first adherent culture for 10 days, the yield of the primary mesenchymal stem cells can be greatly increased in a short time by combining with the second adherent culture, the tissue utilization rate is improved, and the obtained cells have high purity and good differentiation capability; meanwhile, the operation steps are simple and convenient, and the cell collection operation can be completed only by two times.

Drawings

FIG. 1 morphology of isolated primary cells (40X) from day 10 of first adherent (left panel) and day 7 of second adherent (right panel) culture of umbilical cord tissue blocks

FIG. 2 harvesting of Primary cell numbers (left panel) and viability (right panel) after first adherent (1 st) and second adherent (2 nd) culture of umbilical cord tissue blocks

FIG. 3 shows the results of cell surface antigen detection after culture of umbilical cord tissue blocks at first attachment (Panel a) and second attachment (Panel b), harvested cells, and passage to P1

FIG. 4 staining results of adipogenic (400X), chondrogenic (50X), osteogenic (50X) cells after passage to P1 after the harvest of cells after the first and second adherent culture of umbilical cord tissue blocks

Detailed Description

The main steps comprise the following contents

1. Collecting umbilical cord tissues: the collected umbilical cord of the newborn is negative in detection result of virus (infectious diseases such as hepatitis B virus, hepatitis C virus, human immunodeficiency virus, cytomegalovirus, syphilis and the like), has no family history (blood system diseases, metabolic diseases, chromosome abnormality, immunodeficiency disease and the like), is collected to be about 30cm in length, and is stored in physiological saline and transported to a laboratory for treatment.

2. F, gum separation: the method comprises the steps of immediately washing fresh umbilical cord tissue with a large amount of physiological saline after the surface of the umbilical cord tissue is sprayed with 75% ethanol, shearing two ends of the umbilical cord with surgical scissors, draining blood in a blood vessel with forceps, cleaning, shearing the umbilical cord tissue into a tissue section (the edema part is discarded) with the scissors, dividing the umbilical cord tissue into two parts along the artery with the forceps, removing two arteries and one vein, and stripping the Fahrenheit glue with the forceps. Soaking the obtained Fahrenheit gelatin in 6-10 mL of complete culture medium containing 10% platelet lysate for about 30min at room temperature, scraping the culture medium and mucus on the surface of the Fahrenheit gelatin by using forceps, placing the culture medium and mucus in a 50mL centrifuge tube, and cutting the culture medium and mucus into pieces with the diameter of 1-3 mm by using surgical scissors ³ The tissue mass of (1).

3. Tissue block culture: tissue pieces were evenly distributed at the bottom of the T75 flask and incubated at 37 ℃. After 12h of culture, 6mL of complete medium containing 10% serum replacement was added to each T75 flask for culture, after which the medium was changed every 3 to 5 days.

4. And (3) tissue block secondary culture: cell dissociation around the tissue block can be observed under a microscope after three days of culture at the earliest, and the cell fusion degree can reach about 80 percent after 10 days of culture. And (3) collecting the tissue blocks and the culture solution, centrifuging for 5min at 300g, pouring the culture solution, re-inoculating the tissue blocks into a T75 culture flask, and culturing according to the step 3.

5. Collecting primary cells: when the fusion degree of the dissociated cells reaches about 80%, adding 2mL of TrypLE express pancreatin substitute into each culture flask for digestion for 2min, adding 6mL of complete culture medium to stop digestion, collecting liquid, filtering and centrifuging by using a 100-micron filter, discarding supernatant, adding 0.5mL of complete culture medium containing 5% hemoreplacement for heavy suspension, taking 20 mu L of cell suspension, counting by using a CountStar counter, and recording the obtained number and the survival rate of primary cells twice. After the culture is expanded to P1, cell surface antigen detection and trilineage differentiation capacity detection are carried out.

6. The cell surface antigen detection method comprises the following steps: will contain 2.0X 10 ⁶ The cell suspension of P1 umbilical cord mesenchymal stem cells was divided into 9 EP tubes in a volume of 110. Mu.L per tube, and 2.0X 10 cells were placed in each tube ⁵ Individual cells, and labeled as follows: PE-ISO/CD11/CD19/CD34/CD45/HLA-DR/CD73/CD90/CD105, 3000rpm/min, centrifuge for 6min, carefully remove supernatant. Adding 25. Mu.L of PBS for resuspension, adding 5. Mu.L of each of the monoclonal antibodies PE-ISO/CD11/CD19/CD34/CD45/HLA-DR/CD73/CD90/CD105, gently blowing, mixing, and incubating in a refrigerator at 4 ℃ for 30min in the dark. After the incubation was terminated, the sample was washed once with 500. Mu.L PBS per tube at 3000rpm for 6min, and the supernatant was carefully discarded; after repeated washing, 200. Mu.L of PBS was added for resuspension, transferred to a flow tube, and subjected to flow detection using a flow cytometer.

7. And (3) detecting the cell trilinear differentiation capacity: taking P3-substituted umbilical cord mesenchymal stem cells, and adding 1.0 × 10 ⁴ Inoculating at an individual cell/well density into 12-well plates, adding 1ml of complete medium, incubating the cells at 37 deg.C, 5% ₂ The cells were cultured in an incubator until 60% confluency was achieved. The 12-well cells were divided into three groups, and the culture media were replaced with lipid-inducing, osteogenic-inducing and chondrogenic induction media, respectively. The medium was changed at intervals with the same medium. After 14 days of induction, the adipogenic-induced cells were stained with oil red ORed oil drops are observed; inducing for 21 days, carrying out alizarin red staining on cells induced by osteogenesis, and observing calcium nodules; after 19 days of culture, chondrogenic induced cells were stained with an a Li Xinlan stain and the results recorded.

8. The experimental results are as follows: the number of primary cells collected twice from the umbilical cord tissue with the length of about 30cm and the diameter of about 1cm after the operation is 1.06 multiplied by 10^8 in total, the number of the primary cells separated from each centimeter of the umbilical cord tissue reaches 3.53 multiplied by 10^6, the umbilical cord tissue is expanded and cultured to P5 for clinical application, and the umbilical cord tissue with the length of 30cm can be used for more than 1 ten thousand times clinically; and the cell morphology survival rate obtained by planting the tissue blocks twice has no obvious difference, the surface antigen expression and differentiation capacity of the P1 cell have no obvious difference, and the quality stability is ensured. The number of primary cells obtained by the secondary adherence of the tissue block is far lower than that obtained by the primary adherence of the tissue block, which shows that after the two times of planting, most of cells in the tissue block are dissociated to the outside of the tissue, and the utilization rate of the tissue block is improved.

The foregoing examples are intended to further illustrate preferred embodiments of the invention, and are not intended to be exhaustive. Other embodiments of the invention based on the present invention, which can be made by a person skilled in the art without inventive step, belong to the scope of protection of the present invention.

Claims

1. A method for improving the yield of umbilical cord mesenchymal stem cells in a shorter culture time is characterized in that primary mesenchymal stem cells with stable quality for clinical application can be obtained in a large amount, and the method comprises the following steps:

A. collecting an umbilical cord: collecting virus (infectious diseases such as hepatitis B virus, hepatitis C virus, human immunodeficiency virus, cytomegalovirus, syphilis, etc.) with negative detection result, no family history (blood system diseases, metabolic diseases, chromosome abnormality, immunodeficiency disease, etc.), and healthy neonatal umbilical cord tissue;

B. washing the umbilical cord: spraying 75% ethanol on the surface of the whole umbilical cord for 5-10 s, immediately washing the surface of the umbilical cord with a large amount of normal saline, and washing the umbilical cord for three times in a 10cm culture dish filled with 20-25 ml of normal saline;

C. segmenting an umbilical cord: placing the umbilical cord into a new culture dish containing 20-25 mL of physiological saline, cutting off two ends of the umbilical cord by using surgical scissors, draining blood in a blood vessel, cleaning twice by using the saline, and cutting the umbilical cord tissue into tissue sections with the length of 4-5 cm by using the surgical scissors;

D. separating and treating Fahrenheit glue: tearing the umbilical cord along the umbilical vein by using forceps, removing a vein and two arteries, tearing off the amnion, and obtaining the remaining tissue which is the Fahrenheit jelly; putting Fahrenheit into a complete culture medium, soaking for a period of time at a certain temperature, fishing out the Fahrenheit, and shearing the Fahrenheit to 1-3 mm by using surgical scissors ³ Tissue mass;

E. tissue block culture: uniformly spreading the tissue mass at the bottom of the culture flask, and adding CO 5% ₂ Adding a complete culture medium to continue culturing after culturing for a period of time in an incubator at 37 ℃, replacing the complete culture medium once within 3-5 days until the cell fusion degree reaches about 90-95%, separately collecting tissue blocks and cells, carrying out second adherent culture on the tissue blocks until the cell fusion degree reaches about 90-95%, collecting the cells, and combining the primary cells obtained twice.

2. The complete culture medium for Fahrenheit gel immersion as claimed in claim 1 is a mixture of serum replacement and basal medium, wherein the serum replacement is 3-100% by volume and the basal medium is 0-97% by volume.

3. The Fahrenheit gum immersion temperature of claim 1 ranges from 20 to 37 ℃ and the immersion time is from 0.5 to 24 hours.

4. The serum replacement in the complete culture solution according to claim 2, which comprises a reagent having a nutrient component such as human serum replacement, human platelet lysate, or the like.

5. The method of claim 1, wherein the tissue mass is cultured in an incubator for 0-24 hours after the tissue mass is spread evenly on the bottom of the culture flask.