RU2242980C1 - Culture of genetically non-modified human chondroprogenitory cells and method for their preparing - Google Patents

Abstract

FIELD: medicine, traumatology, orthopedics.

SUBSTANCE: invention relates to non-modified animal and human cells and methods for their preparing. The culture of genetically non-modified human chondroprogenitory cells obtained from human embryo hyaline cartilage at stage 1-2 trimesters of pregnancy comprises aggrecan- and collagen II-expressing cells in the amount 80%, not less. Suspension of primary dissociated human chondroprogenitory cells is obtained from human embryo cartilage tissue at stage 1-2 trimesters of pregnancy and cultured in selective cultural medium. Invention provides preparing homogenous cell culture for transplantation, enhanced effectiveness of treatment and avoiding undesirable results.

EFFECT: improved preparing method.

10 cl, 1 ex

Description

The invention relates to undifferentiated animal and human cells and methods for their preparation.

The treatment of degenerative diseases and injuries of the musculoskeletal system is still the most difficult task of medicine. This is due to the widespread prevalence of these diseases, a high percentage of disability, a decrease in the quality of life of patients, and the lack of effective treatment methods. Despite the deepening of ideas about the etiology, pathogenesis, processes of regeneration of bone and cartilage, modern methods common in reconstructive surgery do not always allow to replenish the structure and function of damaged organs and tissues, which is due to degenerative processes in the framework of the disease itself, the massiveness of traumatic damage, the failure of repair systems , due to systemic diseases and age-related characteristics of patients (Mankin et al. 1982., Buckwalter et al. 1994.). One of the points of application in the treatment of diseases of the musculoskeletal system is the stimulation of bone and cartilage tissue repair processes.

Currently, various biological and synthetic materials in the form of implants are widely used, including with the use of auto- and allogeneic fibroblasts, bone marrow mesenchymal stem cells, other elements of the bone marrow, chondrocytes, etc., with the aim of directed osteo- and chondro regeneration (Ashton et al. 1980., Aston et al. 1986., Brittberg et al. 1994.). As shown by several authors, the used biological and synthetic materials do not cause sufficient stimulation of reparative processes to restore the structure and function of the tissue (Coletty et al. 1972., Fumkawa et al. 1980.).

Auto and allogeneic fibroblasts are differentiated cells with low potentials for proliferation, migration, directed differentiation and repair.

Auto- and allogeneic bone marrow obtained by sternal puncture or trypanobiopsy of an adult iliac crest (Yoo et al. 1998.) is characterized by cellular heterogeneity, low hematogenous stem cell counts, which also have limited ability to proliferate, migrate and repair. In addition, fibroblasts, bone marrow cell elements obtained from an adult express histocompatibility antigens of classes I and II. The use of auto- and allogeneic fibroblasts and bone marrow exert a pronounced trophic effect, activating intact tissue sites, however, the recipient does not “develop” in the body and, as a result, is eliminated, but may be rejected and cause local inflammatory reactions with further development not of hyaline, but fibrous-cartilage or fibrous tissue (Coletty et al. 1972, Fumkawa et al. 1980. US Patent Grande et al. 6214369, 04.10. 2001).

Auto- or allogeneic chondrocytes, being mature differentiated cartilage cells, have limited proliferative activity, which does not allow to obtain these cells in sufficient quantities while maintaining the phenotype (Brittberg et al. 1994., Katsube et al. 1999. US McPherson et al. 6150163, 11.21.2000).

Known methods for producing human chondroprogenitor cells from fetal cartilage obtained at different gestation periods include sequential mechanical and enzymatic disaggregation of the original cartilage tissue using various enzyme solutions and their combination (collagenase, pronase, hyaluronidase, deoxyribonuclease, etc.) at various concentrations . Depending on the methods of disaggregation, you can get a different number of cells with the necessary phenotype and a different number of viable cells from 50 to 80%. For the cultivation of chondroprogenitor cells, both Petri dishes uncovered by carriers, culture bottles, and on various non-selective substrates from collagen, agarose, etc. are used (Brittberg et al. 1994, Fuchs et al. 2002). The listed methods of culturing cells in a monolayer do not allow more than 2–3 passages without loss of the phenotype of chondroprogenitor cells that dedifferentiate into fibroblast-like cells. Known methods for the cultivation of chondroprogenitor cells in suspension using various three-dimensional carriers (alginate, polyglycolide, polylactate, atelocollagen), which allows you to conduct culture for a longer time without losing the phenotype of the cells (Kimura et al. 1984., Matsusaki et al. 1998).

Chondroprogenitor cells obtained from fetal cartilage by sequential disaggregation and cultivation in selective media during transplantation proliferate, migrate, integrate into the lesion site, differentiate into chondrocytes or osteocytes, depending on the microenvironment, synthesize the extracellular matrix, stimulate angiogenesis 2160112, 10/12/2000).

The use of a culture of chondro-progenitor cells as a graft can increase the effectiveness of treatment and avoid undesirable results.

An object of the present invention is to obtain a homogeneous culture of chondrogenetic cells for transplantation in traumatic and degenerative diseases of cartilage and bone tissue, to increase the effectiveness of their treatment.

To solve this problem, a group of inventions is proposed, united by a common inventive concept.

One of the objects is the culture of genetically unmodified human chondroprogenitor cells. The culture was obtained from the hyaline cartilage of human embryos of 1-2 trimesters of pregnancy and contains aggrecan and collagen II-expressing cells of at least 80% and not more than 20% of impurity cells. The second object is a method for producing chondroprogenitor cells. Cartilage tissue of human embryos of 1-2 trimesters of pregnancy is washed, mechanically crushed and subjected to enzymatic disaggregation for 40-90 min in a solution containing 0.01-0.1% type II collagenase solution, 0.05-0.1% pronase E. solution. Suspension of primary dissociated chondroprogenitor cells is seeded with a density of at least 1 × 10 ⁶ cells / ml and cultured in a medium containing DMEM / F12 growth medium, 10% fetal calf serum with the addition of growth additives and growth factors.

The environment is repeatedly changed. Upon reaching the cell culture of the confluent state, passaging is carried out using a trypsin solution to disaggregate the monolayer. Sowing density 1 × 10 ⁶ cells / ml.

Cells are cultured for 18-25 days at 37 ° C in an atmosphere of 5% CO ₂ . Passport culture no more than two times.

The method is as follows

For the preparation of a biograft, fetal abortive material is used, obtained by artificially aborting pregnancy in healthy women previously examined for the presence of viral and bacterial infections. Fetal material is transferred into a sterile vessel with a solution of Hanks and gentamicin (80 mg / l). Further work is done under sterile conditions in a laminar box. The cartilaginous tissue extracted with the help of surgical instruments is thoroughly washed from blood clots, and all soft tissues (skin, fascia, muscles, tendons, ligaments) and bone tissue are maximally separated. The resulting cartilage is crushed to pieces of at least 1 mm ³ .

Then subjected to enzymatic and mechanical disaggregation - the release of cellular elements from the tissue matrix.

The disaggregation method consists in using a 0.1% solution of type II collagenase (Sigma), a 0.05% pronase E solution (Sigma), in one-stage incubation of cartilage tissue pieces for 40-90 min in a two-component (1: 1) enzyme solution, followed by grinding by repeated pipetting to obtain a unicellular suspension. The resulting suspension is washed three times by centrifugation at 700 rpm.

Cultivation method

A suspension of primary dissociated cartilage cells obtained from fetal cartilage tissue was cultured on uncovered Petri culture plates in medium containing DMEM + F12 growth medium (1: 1, Gibco BRL), 10% fetal calf serum (NPE PanEco), 1% ITS (insulin, transferrin, selenite. NPE PanEco), 50 μg / ml ascorbic acid (Sigma), 10 μg / ml gentamicin, 5 μg / ml amphotericin B with the addition of growth factors 5-20 ng / ml bFGF (Sigma) and / or 0.1-10 ng / ml TGFβ ₁ (Sigma). The medium is replaced every 3 days. Upon reaching the confluent cell culture, passaging should be performed using a 0.05% trypsin solution to disaggregate the monolayer of the same culture medium, with a culture density of at least 10 × 10 ⁵ cells / ml. It is possible to carry out no more than two passages due to dedifferentiation and a change in the phenotype (reduction in the number of aggrecan and collagen II-expressing cells) of the resulting cells.

Experimentally selected conditions, combining the criteria for selecting the initial biomaterial and the cultivation mode, are optimal to achieve the following results: a system of sequential enzymatic disaggregation of the original tissue allows you to get the maximum number of viable cells of the same phenotype with a small admixture of heterotopic cells; cultivation mode and selective culture media allow to maximize the number of aggrecan and collagen II-expressing cells; cultivation mode and selective culture media make it possible to obtain a homogeneous cell culture containing at least 80% aggrecan and collagen II-expressing cells; cultivation mode and selective culture media allow to obtain a homogeneous cell culture with a content of not more than 20% impurity cells.

The proposed method for the disaggregation and cultivation of chondroprogenitor cells allows you to get the maximum number of homogeneous aggrecan and collagen II-expressing cells with high potentials for proliferation, differentiation and synthesizing the extracellular cartilage matrix. The culture of genetically unmodified chondroprogenitor cells obtained by this method can be used for degenerative diseases and injuries of the musculoskeletal system.

Claims (9)

1. A culture of genetically unmodified human chondroprogenic cells, characterized in that it is obtained from the hyaline cartilage of human embryos of the 1-2 trimester of pregnancy and contains at least 80% aggrecan and collagen II-expressing cells. 2. A method of obtaining a culture of genetically unmodified human chondroprogenic cells, characterized in that the cartilage of a human embryo of the 1-2 trimester of pregnancy is washed, mechanically crushed, then subjected to enzymatic disaggregation by simultaneous incubation in a solution of type II collagenase and pronase E, then the resulting suspension primary dissociated chondroprogenator cells are seeded with a density of at least 1 × 10 ⁶ cells / ml and cultured in a selective culture medium containing growth DMEM + F12 medium, 10% fetal calf serum, growth supplements and growth factors, and when a cell culture reaches a confluent state, passaging is performed. 3. The method according to claim 2, where for the enzymatic disaggregation of cells using a 0.01-0.1% solution of type II collagenase and a 0.05-0.1% solution of pronase E. 4. The method according to claim 2, where a selective culture medium is used with the addition of 10% fetal calf serum. 5. The method according to claim 2, where they use a selective culture medium with the addition of growth factors 5-20 ng / ml bFGF (basic Fibroblast Growth Factor) and / or 0.1-10 ng / ml TGFβ ₁ (Transforming Growth Factor β ₁ ) . 6. The method according to claim 2, where a selective culture medium is used with the addition of growth additives 1% ITS (insulin, transferrin, selenite), 50 μg / ml ascorbic acid. 7. The method of claim 2, wherein said cells are cultured on non-coated Petri culture plates. 8. The method according to claim 2, where the passaging of said cells is carried out no more than two times upon reaching a culture of confluent state. 9. The method according to claim 2, where the said cells are cultured for 18-25 days at a temperature of 37 ° C in an atmosphere of 5% CO ₂ .