RU2710263C1 - Method for producing and culturing fibroblast-like cells from a newborn's umbilical cord - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention refers to medicine and can be used for producing and culturing fibroblast-like cells from a newborn's umbilical cord. That is ensured by umbilical cord sampling in healthy females following the delivery of the cesarean section. Umbilical cord is washed three times with sterile Hanks solution with an antibiotic of blood clots and mucus. Blood vessels are removed. Remaining fabric is ground, material is transferred into centrifuge tubes and poured with 0.2 % collagenase solution. Tubes are placed in a temperature shaker at a temperature of +37 °C, rotation speed 100 rpm for 16 hours. Content is filtered into new test tubes. Sterile 0.02 % Versene solution is introduced into them at ratio of 1:1 and centrifuges are tested in a cold centrifuge for 20 minutes. Cell pellet is resuspended in complete nutrient medium and number of recovered viable cells is counted. Cells are inoculated into a culture flask. Culturing is carried out in CO₂ incubator and medium change every 5 days of cultivation. When cell monolayer confluence reaches 80 %, cells are re-crossed at ratio of 1:2.

EFFECT: invention enables to obtain fibroblast-like cells without the risk of contaminated opportunistic flora.

1 cl, 1 tbl

Description

The invention relates to the field of biotechnology, specifically to the technology of isolation, cultivation of fibroblast-like cells from the umbilical cord.

A known method of producing fibroblasts from the umbilical cord of a newborn, obtained during normal childbirth at 39-40 weeks of gestation [1]. The disadvantages of the method are: the risk of contamination of the donor material with a conditionally pathogenic flora, which is contained in the birth canal, a complex technological process for obtaining a suspension of cells from the donor material.

There is a method of producing fibroblast-like cells from the umbilical cord of a newborn, obtained during normal delivery at 39-40 weeks of gestation [2]. After washing the donor material from blood and mucus, manually cut the umbilical cord into fragments, rinse them again with Hanks solution, grind the fragments with a homogenizer to a homogeneous mass; carry out enzymatic treatment using 0.075% type I collagenase and type IV collagenase (ratio of enzyme concentrations 1: 1 in a volume ratio of 1: 5) for 8-10 hours at 37 ° and periodically stirring the contents of the tubes; pass the obtained substrate into a new centrifuge tube through a double sieve for cells; centrifuged for 8 minutes at 1000 rpm./min; a pellet of fibroblast-like cells is obtained, which are resuspended in the culture medium with the addition of a basic fibroblast growth factor; calculate the total number of cells obtained in the Goryaev chamber, introduce cells into Petri dishes at a seed dose of 8-15 × 10 ³ / cm ² ; upon reaching 70-80% confluence of the monolayer, the cells are subcultured, cultured until passage 4-6, while the cells retain their diploid karyotype and high proliferative activity. This method is taken as a prototype.

The disadvantages of the prototype are: the risk of contamination of the donor material by conditionally pathogenic flora, which is contained in the birth canal, the complex technological process of obtaining a suspension of cells using manual labor; the use of several types of enzymes in the processing of the umbilical cord; insufficient inactivation of enzymes during washing; calculation of the inoculative dose based on the total number of cells received, and not on viable cells.

The aim of the invention is to provide a method for producing and culturing fibroblast-like cells from the umbilical cord of a newborn.

This goal is achieved by the fact that the umbilical cord is taken in healthy women after giving birth by cesarean section, the umbilical cord is washed three times with sterile Hanks solution with an antibiotic from blood clots and mucus, blood vessels are removed, the remaining tissue is crushed using a laboratory mixer; transfer the material to centrifuge tubes and pour 0.2% solution of collagenase from hepatopancreas crab BioloT LLC in a ratio of 1: 1; the tubes are placed in a thermo-shaker at a temperature of + 37 ° C, a rotation speed of 100 rpm for 16 hours; filter the contents into new tubes through cell filters with a pore size of 70 microns; contribute sterile 0.02% Versen's solution in a ratio of 1: 1; the tubes are centrifuged in a cold centrifuge at a temperature of + 4 ° C, a rotation speed of 1200 rpm for 20 minutes; the supernatant is disposed of; the cell pellet was resuspended in complete culture medium, including αMEM with glutamine, 10% FBS, 8 μg / ml gentamicin; count the number of allocated viable cells in the Goryaev chamber using a 0.4% trypan blue solution; seeded cells at a dose of 1 × 10 ⁵ viable cells / cm ² in a culture bottle with a similar complete nutrient medium; cultivation is carried out in a CO ₂ incubator at a temperature of + 37 ° C, 5% CO ₂ and constant humidity; a change of medium is carried out every 5 days of cultivation, while the conditioned culture medium from the bottle with cells is completely taken with a pipette; centrifuge it in a cold centrifuge at a temperature of + 4 ° C, a rotation speed of 1500 rpm for 10 minutes, after which it is returned to the culture bottle and fresh medium is added in a 1: 1 ratio; upon reaching 80% confluence of the cell monolayer, the cells are reseeded at a rate of 1: 2.

Umbilical cord sampling in examined women after giving birth by cesarean section is carried out in order to exclude contamination of the donor material by conditionally pathogenic flora, which is found in the birth canal. A laboratory mixer is used in the process of grinding donor material in order to exclude the laborious manual process of grinding the umbilical cord. 0.2% of collagenase from crab hepatopancreas of BioloT LLC is used in the process of enzymatic processing of the umbilical cord because it has high collagenolytic activity (up to 750 U / mg according to Mandl) and is not inferior to several enzymes in its action.

In the process of enzymatic processing of tissues, a thermal shaker is used to ensure that it passes with constant uniform mixing of the medium and better interaction of tissues with collagenase, while creating optimal conditions for the release of cells. In addition, the use of a thermal shaker eliminates manual labor in mixing the medium. Cold centrifugation allows you to maintain the set temperature and does not have a damaging effect on the cells. Counting the obtained cells in the Goryaev chamber is carried out in order to calculate the seed dose of viable cells for inclusion in the culture vial.

The conditioned medium is used for reseeding during the cultivation process, adding it to fresh because it contains physiologically active waste products of the cells cultivated in it, this medium optimizes cell proliferation in the early stages of cultivation, and ensures the accumulation of cells in the required quantity.

The method is as follows. The umbilical cord is taken in healthy women after giving birth by cesarean section. The umbilical cord is washed three times with a sterile Hanks solution with an antibiotic from blood clots and mucus, blood vessels are removed, the remaining tissue is crushed into fragments using a laboratory mixer. The crushed tissue is transferred to centrifuge tubes and poured with a 0.2% solution of collagenase from hepatopancreas crab of BioloT LLC in a ratio of 1: 1. The tubes are placed in a thermo-shaker at a temperature of + 37 ° C, a rotation speed of 100 rpm for 16 hours. Filter the contents into new tubes through cell filters with a pore size of 70 μm. A sterile 0.02% Versen solution in a ratio of 1: 1 is introduced into them. The tubes are centrifuged in a cold centrifuge at a temperature of + 4 ° C and a rotation speed of 1200 rpm for 20 minutes. The supernatant is disposed of. The cell pellet was resuspended in complete culture medium, including αMEM with glutamine, 10% FBS, 8 μg / ml gentamicin. Count the number of allocated viable cells in the Goryaev chamber using a 0.4% trypan blue solution. Sow cells at a dose of 1 × 105 viable cells / cm2 in a culture vial with a similar complete nutrient medium. Cultivation is carried out in a CO ₂ incubator at a temperature of + 37 ° C, 5% CO ₂ and constant humidity. A change of medium is carried out every 5 days of cultivation, while the conditioned culture medium from the vial with cells is completely removed with a pipette. This medium is centrifuged in a cold centrifuge at a temperature of + 4 ° C, a rotation speed of 1500 rpm for 10 minutes, after which it is returned to the culture bottle and fresh fresh similar medium is added in a 1: 1 ratio. Upon reaching 80% confluence of the cell monolayer, the cells are reseeded at a rate of 1: 2.

The method is illustrated by a clinical example. In healthy women, umbilical cord fragments (7-10 cm) were taken after cesarean section after giving birth. Obtaining and culturing fibroblast-like cells from donor material was performed according to the developed method. The characteristics of the obtained fibroblast-like cell lines are shown in Table 1. The cells had a high adhesion index (88 ± 0.2% - 90.5 ± 0.5%), a relatively similar proliferation index (1.5 ± 0.3), and the percentage of viable cells in the culture was from 89.5 to 97.8%. Cells were used to create cell-tissue transplants.

The method of obtaining and culturing fibroblast-like cells from the umbilical cord of newborns can be used in specialized laboratory cell cultures to obtain cell products for regenerative medicine: for research purposes for in vitro testing.

Information sources

1. Patent RU 22308957. dated 10.27.2007 “A method for producing a drug for mesotherapy and a drug obtained in this way” K. Yarygin

2. Patent RU 2384618 dated 03/27/2008 "A method for producing fibroblast-like cells from the umbilical cord of a newborn" Saburina I.N., Isaev A.A., Kiselev S.L., Lagarkova M.A., Kosheleva N.V., Melikhova BC , Prikhodko A.V.

Claims (1)

A method for producing and culturing fibroblast-like cells from a newborn's umbilical cord, comprising: taking and preparing an umbilical cord; its mechanical grinding; enzymatic treatment; isolation of a cell suspension as a result of filtration and centrifugation processes, characterized in that the umbilical cord is taken in healthy women after giving birth by cesarean section; the umbilical cord is washed three times with a sterile Hanks solution with an antibiotic from blood clots and mucus, blood vessels are removed, the remaining tissue is crushed using a laboratory mixer; transfer the material to centrifuge tubes and pour 0.2% solution of collagenase from hepatopancreas crab BioloT LLC in a ratio of 1: 1; the tubes are placed in a thermo-shaker at a temperature of + 37 ° С, rotation speed of 100 rpm for 16 hours; filter the contents into new tubes through cell filters with a pore size of 70 microns; contribute sterile 0.02% Versen's solution in a ratio of 1: 1; test tubes are centrifuged in a cold centrifuge at a temperature of + 4 ° С, rotation speed of 1200 rpm for 20 minutes; the supernatant is disposed of; the cell pellet was resuspended in complete culture medium, including αMEM with glutamine, 10% FBS, 8 μg / ml gentamicin; count the number of allocated viable cells in the Goryaev chamber using a 0.4% trypan blue solution; seeded cells at a dose of 1 × 10 ⁵ viable cells / cm ² in a culture bottle with a similar complete nutrient medium; cultivation is carried out in a CO ₂ incubator at a temperature of + 37 ° C, 5% CO ₂ and constant humidity; a change of medium is carried out every 5 days of cultivation, while the conditioned culture medium from the bottle with cells is completely taken with a pipette; centrifuge it in a cold centrifuge at a temperature of + 4 ° C, rotation speed of 1500 rpm for 10 minutes, then return to the culture bottle and add fresh medium in a ratio of 1: 1; upon reaching 80% confluence of the cell monolayer, the cells are reseeded at the rate of 1: 2.