RU2620981C2 - Method of obtaining the culture of mesenchymal human stem cells from the perivascular space of the vein of umbilical cord - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: method involves cutting out the umbilical cord fragment, filling it with 0.2% collagenase solution, closing its both ends and holding for 20-40 minutes at 37°C, followed by washing with a balanced saline solution, filling a new portion with a 0.2% collagenase solution, closing both ends and maintaining for 30-60 minutes at 37°C. After the second holding, the said umbilical vein is washed with balanced saline solution, and obtained by leaching liquid is centrifuged, and the obtained pellet is cultivated on selective mediums.

EFFECT: invention allows to increase the yield and purity of obtained culture of human mesenchymal stem cells and in some cases to reduce the time of obtaining human mesenchymal stem cells.

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Description

The invention relates to the field of cellular biology and biotechnology, in particular to cellular technologies, in particular to methods for producing human mesenchymal stem cells used in regenerative therapy.

Mesenchymal stromal (stem) cells (MSCs) are multipotent cells originating from the mesoderm germ layer, which are capable of self-maintaining the population and differentiating into mesenchymal cells: chondrocytes, osteocytes, adipocytes. There are methods for the differentiation of MSCs into cells that are formed in the body from other germ layers: epithelial cells, neuron-like cells. MSCs have an immunomodulatory effect, secrete angiogenic factors, and are also capable of migration to the site of damage. In addition, MSCs are able to differentiate into cells of damaged tissues, thus contributing to the restoration of the functional integrity of an organ or tissue. Such properties make it possible to consider MSCs as a promising tool for regenerative therapy based on cell technologies.

The umbilical cord, in particular the Wharton jelly and the perivascular space of the vessels, as well as the umbilical cord blood and placenta, which are usually disposed of after delivery, are convenient sources of MSCs, since they contain a large amount of MSCs in terms of 1 gram of tissue, and the amount of tissue obtained is high in compared to other sources. In addition, cells from these sources have a higher proliferative potential compared with adult MSCs in adipose tissue and bone marrow, which allows to reduce the expansion time, that is, cell growth without loss of differentiation potential in vitro. Such expansion is necessary in most cases, because for the majority of therapeutic and surgical procedures using MSCs, no source of MSC production allows obtaining cells in an amount sufficient for an adult patient, i.e. 20-80 million, depending on the patient’s body weight and the procedure.

Five zones were found in the umbilical cord, from which it is possible to isolate MSCs:

1. 20-50% of the freshly isolated mononuclear fraction of cord blood;

2. subendothelial layer of the umbilical vein;

3. the outer layers (perivascular space) of the umbilical cord vessels;

4. intravascular space;

5. subamniotic space.

MSCs isolated from different zones of the umbilical cord are separate populations with the same immunophenotype, but different proliferative activity and differentiating ability, a review of these data was made in the article: Conconi M.T., Di Liddo R., Tommasini M., Calore C., Parnigotto PP Phenotype and differentiation potential of stromal populations obtained from various zones of human umbilical cord: an overview // The Open Tissue Eng Regen Med J. - 2011. - V. 4. - P. 6-20. MSCs of the perivascular space are characterized by a high in vitro population growth rate and a high level of expression of pancytokeratin and CD146 [Carvalho M., Teixeira F.G., Reis R.L., Sousa N., Salgado AJ. Mesenchymal stem cells in the umbilical cord: phenotypic characterization, secretome and applications in central nervous system regenerative medicine // Current stem cell research & therapy. - 2011. - V. 6. - No. 3. - P. 221-228]. In addition, MSCs from these two regions differ in the level of production of IL-6 and Flt3 [Xu M., Zhang B., Liu Y., Zhang J., Sheng H., Shi R., Chen H. The immunologic and hematopoietic profiles of mesenchymal stem cells derived from different sections of human umbilical cord // Acta biochimica et biophysica Sinica. - 2014. - C. 100].

In the article: Ennis J., Sarugaser R., Gomez A., Baksh D., Davies J.E. Isolation, characterization, and differentiation of human umbilical cord perivascular cells (HUCPVCs) // Methods in cell biology. - 2008. - V. 86. - P. 121-136 shows the possibility of obtaining MSCs from the perivascular space of the umbilical vein.

A known method of obtaining a culture of MSCs from the perivascular space of the umbilical cord described in: Sarugaser R., Lickorish D., Baksh D., Hosseini M.M., Davies J.E. Human umbilical cord perivascular (HUCPV) cells: a source of mesenchymal progenitors // Stem Cells. - 2005. - V. 23. - No. 2. Z. 220-229. The method consists in the fact that the vessels - the vein and both arteries - of the umbilical cord are alternately cut out of the Warton jelly, the ends of each vessel are sutured together to form a closed loop, and incubated in a 0.1% collagenase solution for 18-24 hours. Then the loops are removed, the solution is centrifuged, the precipitate is washed several times and cultured in the standard way. The above method is very time-consuming, includes complex procedures for the preparation of vessels from the Warton jelly and a long 24-hour enzymatic treatment. This makes the above method unsuitable for therapeutic use for a large number of patients, especially when it is necessary to quickly obtain MSCs. The viability of the isolated cells is low as a result of prolonged 24-hour enzymatic treatment.

In the article: Aizenshtadt A.A., Ivanova N.A., Bagaeva V.V., Smolyaninov A.B., Samoilovich M.P., Klimovich V.B. Intracellular immunoglobulins in the Namalva and U266 lines upon co-cultivation with mesenchymal cells // Cytology - 2014. - V. 56. - No. 2. - P. 117-121 describes a method for obtaining MSCs from the perivascular space of the umbilical cord, which allows to reduce the processing time of the umbilical cord from 16-26 hours to 1.5 hours. This method consists in the fact that the umbilical vein is a whole unground crushed fragment of the cord (10- 30 cm), a 0.2% type IV collagenase solution is introduced, closed at both ends with terminals and incubated for 60 minutes at a temperature of 37 ° C. After incubation, the umbilical vein is washed with phosphate-buffered saline and the liquid obtained by washing is centrifuged. The precipitate was transferred to a selective medium and several passages were cultured in culture containers adapted for culturing adhesive cells.

The obtained cells have an immunophenotype of MSCs and are able to differentiate in osteo-, chondro- and adipogenic directions. The above method significantly reduces the time to obtain a culture of MSCs by eliminating the stage of preparation of the umbilical cord vessels and reducing the time of collagenase treatment. However, the disadvantages of the method are: a) low cell yield - 1 million cells per 10 cm of the length of the cord after the first passage; b) contamination of MSC secreted by cells of other types, mainly endotheliocytes; c) low safety of MSCs as a result of prolonged 1-hour incubation in a collagenase solution, and as a result, their low proliferative activity; d) a large time of MSC explantation in culture (5 days). These disadvantages make the above method unsuitable for commercial use. This method is the closest to the claimed invention and is selected as a prototype.

The objective of the present invention is to provide a method for producing a culture of MSCs from the perivascular space of the umbilical cord, which reduces the time to obtain a culture of MSCs, increases the yield and safety of the resulting cells, increases the purity of the culture of MSCs.

The problem is solved in that in a method of obtaining a culture of MSCs from a perivascular space of the umbilical cord, a fragment of the umbilical cord is cut out, the vein of the mentioned cord is filled with a solution of 0.2% collagenase, both ends of the umbilical cord are closed and incubated for at least 20 minutes, but no more than 40 minutes at a temperature of 37 ° C, after which the aforementioned vein is washed with a balanced saline solution, the vein is filled with a new portion of the aforementioned collagenase solution, both ends are closed of said umbilical cord and held for at least 30 minutes but not more than 60 minutes at a temperature of 37 ° C, then washed vein said balanced salt solution obtained by washing the centrifuged liquid. The resulting precipitate is cultured in a known manner using selective media.

Unlike the prototype, in the inventive method for producing a culture of MSCs, two incubation cycles in a collagenase solution are carried out. After the first incubation of the umbilical cord in a 0.2% solution of collagenase, which fills only the lumen of the umbilical vein, part of the extracellular matrix of the wall of the umbilical vein is destroyed. Thus, the solution collected from the umbilical vein contains mainly cells from zones 1-2. That is, in addition to MSCs, a significant number of endotheliocytes, blood cells, hematopoietic stem cells, etc. are found in this portion. After the second incubation, the obtained fraction contains mainly cells of 3-4 zones, and the Warton jelly mucopolysaccharides (zone 5) are most often absent. As a result of subsequent washing, excess collagenase is removed, thus, most of the cells are preserved from its damaging effects. When implementing this method, the MSC fraction in the preparation of isolated cells increases, the total time for obtaining a cell culture decreases, the cell safety and cell culture purity increase, the proportion of actively proliferating MSCs of the perivascular space increases, and the percentage of slowly growing Warton jelly MSCs decreases, up to their complete absence. MSCs of the culture obtained by the implementation of this method retain their viability and proliferative potential.

If necessary, both fractions after the first and second treatment with collagenases are cultivated separately on selective media using adhesive plastic, and the resulting MSC cultures are combined for use in regenerative therapy.

Described in the inventive method, the sequence of operations of processing the obtained fragment of the umbilical cord, the selected modes and substances used in this case, provide a solution to the problem.

The invention is illustrated by the following graphic materials.

In FIG. 1 shows the number of MSCs and cells of other types obtained after the first passage, depending on the duration of the first and second exposure in a 0.2% collagenase solution.

In FIG. 1a shows the number of MSCs and other types of cells obtained by the method described in example 1.

In FIG. 1b shows the number of MSCs and other types of cells obtained by the method described in example 2.

In FIG. 1c shows the number of MSCs and other types of cells obtained by the method described in example 3.

In FIG. 2 shows microscope images of cell cultures obtained with different durations of the first and second exposure in a 0.2% collagenase solution.

In FIG. 2a shows a microscope image of a cell culture obtained after first exposure to a 0.2% collagenase solution for 20 minutes, as described in example 1.

In FIG. 2b shows a microscope image of a cell culture obtained after a second exposure in a 0.2% collagenase solution for 30 minutes, as described in example 1.

In FIG. 2c shows a microscope image of a cell culture obtained after first exposure to a 0.2% collagenase solution for 30 minutes, as described in example 2.

In FIG. 2g shows a microscope image of a cell culture obtained after a second exposure in a 0.2% collagenase solution for 45 minutes, as described in example 2.

In FIG. 2e shows a microscopic image of a cell culture obtained after first exposure to a 0.2% collagenase solution for 40 minutes, as described in Example 3.

In FIG. 2e shows a microscope image of a cell culture obtained after a second exposure in a 0.2% collagenase solution for 60 minutes, as described in example 3.

Images were obtained using a biological microscope for laboratory research (Axiovert 40 series microscope with an Axiovert 40 CFL tripod) and a microscope camera (Progress ST 3 Carl Zeiss, Germany).

The method was carried out as follows.

Example 1. Obtained in a standard way, the umbilical cord was placed in a vessel containing a sufficient amount of “heparin water” (5 ml of heparin per 200 ml of saline), with the addition of an antibiotic, after which it was transported to the laboratory. The umbilical vein was thoroughly washed with a balanced saline solution, such as Versen's solution, using a syringe. The umbilical vein was then washed with a 0.2% solution of collagenase type I and IV (1: 1) in PBS using a syringe. The umbilical cord was terminated on one side, then the umbilical vein was filled with the aforementioned collagenase solution. We clamped the umbilical cord on the opposite side. The clematized sample was placed in a Petri dish and kept in a shaker-incubator, for example, ES-20 (Biosan, Latvia), at a temperature of 37 ° C and gentle stirring for 20 minutes. After this, the collagenase solution was collected and the umbilical vein was thoroughly washed with a balanced saline solution, for example, PBS solution. The collagenase solution and saline after washing were collected and centrifuged, for example, in a SM-6M centrifuge (Elmi, Latvia), 7 minutes at 1100 rpm. The supernatant was removed, the pellet was placed in a 75 cm ² culture vial, to which 10-15 ml of the complete culture medium was previously added, for example, MEM-α / Advanced Stem Cell Media (HyClone, Germany) supplemented with 20% fetal bovine serum for MSC or Serum Substitute Advanced Supplement for Stem Cells (HyClone, Germany). The vials were placed in a CO ₂ incubator, for example, BBD 6220 (Thermo, Germany), to attach the MSC fraction to the plastic; the exposure time was 3-5 days, depending on the state of the cells. The obtained first MSC fraction was used for comparison. The umbilical vein was washed a second time with said collagenase solution using a syringe. The umbilical cord was terminated on one side, then the umbilical vein was filled with the aforementioned collagenase solution. We clamped the umbilical cord on the opposite side. The clemented sample was placed in a Petri dish and kept in the said shaker-incubator at a temperature of 37 ° C and gentle stirring for 30 minutes. After standing, said collagenase solution was collected and the umbilical vein was thoroughly washed with said balanced saline solution. The collagenase solution and saline after washing were collected and centrifuged on the centrifuge for 7 minutes at 1100 rpm. The supernatant was removed, the pellet was placed in a 75 cm ² culture vial, to which 10-15 ml of the complete culture medium was previously added. The vials were placed in the aforementioned CO ₂ incubator to attach the MSC fraction to the plastic; the exposure time was 3-5 days, depending on the state of the cells. Next, MSCs were cultured by a known method of culturing MSCs.

Example 2. The preparation of the MSC culture was carried out according to the scheme shown in example No. 1, characterized in that the first exposure in the said collagenase solution was carried out for 30 minutes, and the second aging in the said collagenase solution was carried out for 45 minutes.

Example 3. Obtaining a culture of MSCs was carried out according to the scheme shown in example No. 1, characterized in that the first exposure in the aforementioned collagenase solution was carried out for 40 minutes, and the second aging in the aforementioned collagenase solution was carried out for 60 minutes.

The applicant asks to consider the submitted application materials "A method of obtaining a culture of human mesenchymal stem cells from the perivascular space of the umbilical cord vein" for the grant of a patent of the Russian Federation for the invention.

Claims (2)

1. A method of obtaining a culture of human mesenchymal stem cells from the perivascular space of the umbilical cord, which consists in cutting out a fragment of the umbilical cord, filling the vein of the mentioned fragment of the umbilical cord with a 0.2% collagenase solution, closing both ends of the mentioned fragment of the umbilical cord and keeping it for at least 20 minutes, but not more than 40 minutes at a temperature of 37 ° C, characterized in that after that the said vein is washed with a balanced saline solution, filled with a new portion of said collagenase solution, cover both ends of said umbilical cord and incubated for at least 30 minutes, but not more than 60 minutes at 37 ° C, after which said vein is washed with a balanced saline solution, the liquid obtained by washing is centrifuged, the resulting precipitate is cultured on selective environments. 2. A method of obtaining a culture of human mesenchymal stem cells from the perivascular space of the umbilical cord according to claim 1, characterized in that the liquid obtained by washing after the first incubation cycle in a 0.2% collagenase solution is collected, centrifuged, the resulting precipitate is cultured on selective media and the resulting cell culture is added to the culture obtained according to claim 1

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