RU2285039C2 - Method for preparing donor chondroblasts - Google Patents

Abstract

FIELD: biotechnology, traumatology, orthopedics.

SUBSTANCE: invention relates to a method for preparing donor chondroblasts. Chondroblasts are isolated from vertebra body growth plates of mini pig of age before 4 months. Method involves enzymatic treatment of milled donor tissue with collagenase taken in the concentration 1.5% for 8 h in 10-fold volume above tissue to be treated. Prepared chondroblasts are cultured in the DMEM cultural medium in the presence of 20% of FBS and the following reinoculation 2 times per a week with 1/(2-3) ratio of inoculation. Invention provides preparing the necessary material used in correction of degenerative-destructive changes in cartilage tissue in minimal consumptions.

EFFECT: improved preparing method.

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Description

The invention relates to biotechnology, namely traumatology and orthopedics, and can be used to increase the number of specialized cells promising for the correction of degenerative-destructive changes in cartilage tissue.

Known methods of culturing cells derived from articular cartilage.

Cultivation of articular chondrocytes is performed on the following culture media: DMEM culture medium supplemented with 10% cow fetal serum (FBS), ascorbic acid (50 μg / ml) [Weisser J., Rahfoth B., Timmermann A., Aigner T., Brauer R . and von der Mark K. Role of growth factors in rabbit articular cartilage repair by chondrocytes in agarose. // Osteoarthritis and Cartilage. 2001. No. 9. Supplement A. P.48-54], nutrient medium F12 with 10% FBS and ascorbic acid [Badger A. M., Roshak A. K., Cook. MN, Newman - Tarr T. M., Swift B. A., Carlson K., Connor JR, Lee J. C., Gowen M., Lark MW and Kumar S. Differential effects of SB 242235, a selective p38 mitogen- activated protein kinase inhibitor, on IL-1 treated bovine and human cartilage / chondrocyte cultures. // J. Osteoarthritis and Cartilage. 2000. No8. P. 434 - 443], DMEM / P12 medium or RPMI / NCTC medium (1/1) [Freyria A.-M., Cortial D., Ronziere M.-C., Guerret S., Herbage D. Influence of medium composition, static and frozen conditions on the proliferation of and matrixprotein expression of bovine articular chondrocytes cultured in a 3-D collagen scaffold. // Biomaterials 2004. No. 25. P.687-697] with the addition of 10% serum. FBS induces a dose-dependent effect on chondrocyte proliferation with a maximum at a serum concentration of 20% [Guerne P, Sublet A, Lotz M. Growth factor responsiveness of human articular chondrocytes: distinct profiles in primary chondrocytes, subcultured chondrocytes and fibroblasts. // J. Cell Physiol. 1994. No. 158. P.476-84]. It is possible to cultivate chondrocytes in serum-free medium supplemented with 1% ITS, 1 mM cysteine and 1 mM pyruvate [Freyria A.-M., Cortial D., Ronziere M.-C., Guerret S., Herbage D. Influence of medium composition, static and frozen conditions on the proliferation of and matrixprotein expression of bovine articular chondrocytes cultured in a 3-D collagen scaffold. // Biomaterials 2004. No. 25. P.687-697]. All nutrient media include ascorbic acid. Adding ascorbic acid to the nutrient medium can provoke bacterial infection of the cell culture and change its properties.

Closest to the claimed method is a method of culturing chondroblasts from intervertebral discs [Masuda K., Sah R.L., Hejna M.J., Thonar E.J.-M.A. A novel two-step method for the formation of tissue-engineered cartilage by mature bovine chondrocytes: the alginate-recovered-chondrocyte (ARC) method. // J. of Orthopedic Research. 2003. No.21. P.39-148] using DMEM growth medium supplemented with 6% FBS, 50 μg / ml ascorbic acid and 100-150 mmol / L sucrose. A change of the nutrient medium is performed every 2 days. The number of passages does not exceed 5 times. The main disadvantages of this method of cultivation are the use of a complex nutrient medium for cultivation and the frequent change of the nutrient medium, which can lead to contamination of the cell culture with various infectious agents; the cultivation of different parts of the disk separately, which with further use of the culture can lead to the formation of fibroblast-like cells.

The objective of the invention is to propose a method for the cultivation of chondroblasts of growth plates of vertebral bodies, promising for use in cell therapy for the correction of degenerative-destructive changes in cartilage tissue.

The solution of the problem allows to achieve a positive therapeutic effect.

The planned results will be used in surgical practice. The use of donor material for the correction of degenerative and disruptive changes in the cartilage tissue will normalize the structure of the cartilage tissue, reduce the postoperative rehabilitation period and the number of postoperative hospital days, and improve the patient’s quality of life.

The technical result is achieved due to the fact that for the production of donor chondroblasts, growth plates (PR) of the vertebral bodies of mini-piglets under the age of 4 months are used, since morphohistochemical and biochemical data showed a similarity between PR bodies of the vertebral bodies of mini-pigs (mini-prosenka) and PR of human vertebral bodies. To obtain donor chondroblasts, the vertebral body PRs are used, and not the intervertebral disc tissue (MTD), since it is precisely the vertebral body PRs that, in contrast to the MTD, contain only chondroblasts. The cultivation of cells from MTD can form a fibroblast-like cell type. The maximum yield of chondroblasts from the PR tissue of the vertebral body is ensured by treatment with a 1.5% collagenase solution for 8 hours, and the volume of the collagenase solution should exceed at least 10 times the volume of the treated tissue. The cultivation of the obtained chondroblasts is carried out in DMEM nutrient medium with the addition of 20% FBS, since FBS induces a dose-dependent effect on the proliferation of chondrocytes with a maximum at a serum concentration of 20%. Reseeding of chondroblasts is carried out twice a week, since a frequent change in the nutrient medium can lead to contamination of the cell culture with a variety of infectious agents. Reseeding of chondroblasts was carried out with a seeding rate of 1 / (2-3), since the number of cells during the cultivation period doubles, triple. The number of passages should not exceed 5 times, since at this passage there is a differentiation of chondroblasts into fibroblasts.

The problem is solved due to the fact that they use chondroblasts of the PR of the vertebral bodies of mini-piglets under the age of 4 months; chondroblasts are isolated with a solution of 1.5% collagenase for 8 hours, the volume of the solution exceeds 10 times the volume of the treated tissue; chondroblasts are cultured in DMEM culture medium supplemented with 20% FBS; chondroblasts are subcultured 2 times a week with a screening rate of 1 / (2-3).

The method is as follows.

1. Obtaining PR cells of vertebral bodies of a mini-piglet.

PR vertebral bodies are isolated sterile under aseptic conditions and placed in a Hanks solution with kanamycin 1 g / l for 15 minutes. This time period is chosen empirically, since antibiotics have a toxic effect on a living cell, and the exposure time with an antibiotic should be optimal for maintaining chondroblast viability and exposure to pathogenic flora. After processing the PR, the vertebral bodies are cleaned of soft tissues using a scalpel and ground in a Petri dish with a minimum volume of RPMI medium (so that the chondroblasts remain viable) to sizes of 1-2 mm ³ (optimal size for penetration and action of the enzyme). The crushed cartilage is placed in a solution of 1.5% collagenase (imperially selected for maximum cell output) in a siliconized dish (silicone dish - a commercial product is used so that the cells do not adhere to the wall of the dish), the volume of the solution must exceed the volume of the tissue by at least 10 times; incubated on a shaker at a temperature of 37 ° C (temperature mode of action of collagenase) for 8 hours (the optimal time period of action of collagenase to obtain the maximum number of cells). The suspension is passed through a nylon filter (to separate insoluble cartilage from the debris) and centrifuged for 10 minutes at 1500 rpm. After centrifugation, the cells are resuspended in DMEM growth medium and the concentration of cells in suspension is adjusted taking into account their viability and initial concentration. To do this, stain the cell suspension with an intravital dye, trypan blue. Dead cells are stained intensely blue, in living cells the cell membrane is slightly stained, the dye does not penetrate into the cell.

First, the number of dead cells is counted (under a microscope), then the number of living cells and, finally, the number of all cells. The number of all cells is taken as one hundred percent, the percentage of living cells (viability) is calculated. For sieving, only the number of living cells is taken into account; dead cells are not taken into account.

2. The cultivation of PR cells of the vertebral bodies of the mini-pig.

Cell cultivation is carried out in DMEM nutrient medium supplemented with 20% FBS. After counting viable cells in suspension, the cell concentration is adjusted to 100 thousand cells per ml and sown in culture bottles. Vials are placed in a thermostat at 37 ° C. After 3-4 days, the cells form a monolayer of isocircular and oval cells (Fig.1, 2). After receiving a monolayer, the cells are removed from the surface with a mixture of trypsin solutions of 0.02% and versene 0.025% (for the dissolution and functioning of trypsin). The cell concentration in the Goryaev chamber is calculated. For this, all cells are counted in the "large" squares of the Goryaev chamber, summarized, the resulting number is divided by 60, the resulting number is millions of cells in ml. And spend sieving cells 1 / (2-3). The number of passages should not exceed 5 times in order to avoid the differentiation of chondroblasts into fibroblasts.

Claims (1)

A method of producing donor chondroblasts by isolating and grinding cartilage, carrying out enzymatic processing, filtering and centrifuging the resulting suspension, resuspending chondroblasts and culturing them in DMEM supplemented with 20% FBS, characterized in that mini-vertebral growth plates are used to isolate chondroblasts pigs under the age of 4 months, carry out enzymatic treatment with 1.5% collagenase for 8 hours, in a volume 10 times the volume of the treated tissue, subcultured x ondroblasts 2 times a week with a screening rate of 1 / (2-3).

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