RU2370771C2 - Method of osteogenic potential assessment for mesenchymal stromal cells - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention claims method of osteogenic potential assessment for mesenchymal stromal cells (MSC). Method is based on optic density measurement of MSC cultures within twice increasing cell concentration range, further cell cultivation in osteogenic induction medium and calcium deposit colouring by silver nitrate.

EFFECT: defining optimal conditions of MSC function depending on tissue source type of MSC, MSC cell density, multiplication factor, MSC subculture duration in vitro, serum growth factor concentration in microenvironment, concomitant pathology character.

4 ex, 5 tbl, 4 cl

Description

The invention relates to medicine and cell biology and can be used in transplantology, traumatology and orthopedics for a preliminary assessment in vitro of the functional activity of mesenchymal stromal cells in order to determine their osteogenic potential.

Mesenchymal stromal cells (MSCs) belong to the class of somatic polypotent progenitor cells, which are characterized by the ability to self-maintain and differentiate into different types of mesoderm cells (osteoblasts, chondrocytes, adipocytes). MSCs were first detected in the bone marrow, however, it has now been shown that MSCs can also be isolated from other sources (adipose tissue, placenta, umbilical cord blood, amniotic fluid, cartilage and muscle tissue). In 2006, the Stem Cell Committee of the International Society of Cell Therapy held a conciliation conference and determined the “minimum” criteria specific for MSCs: a) plastic adhesion and fibroblast-like morphology, b) characteristic immunophenotype (expression of CD73, CD90, CD105 and lack of expression of CD34 , CD45, HLA-DR) and c) the ability to differentiate in osteogenic, adipogenic and chondrogenic directions [1].

In modern transplantology, new approaches are widely developed for the use of MSCs, for example, for the restoration of reparative osteogenesis in patients with various disorders of both local and systemic nature [2, 3]. In this case, the use of autologous cellular material is considered the safest, which eliminates hetero- or allotransplantations associated with the solution of histocompatibility problems, ethical and legal issues, and also eliminates the risk of iatrogenic infection.

However, the use of MSCs for autotransplantation necessitates a preliminary assessment of their differentiation potential. The ability of MSCs to directed differentiation in vitro is a key parameter that reflects the functional activity of MSCs. Obviously, the effectiveness of treatment using MSC transplantation will be largely determined by the preservation of their differentiation potentials, which, in turn, directly depends on many factors (age and genetic characteristics of the patient, the nature / prevalence of the pathological process, medication, source of production and features of isolation / expansion of MSCs in vitro, etc.).

The differentiation of MSCs in the osteogenic direction is stimulated by culturing them for 18-21 days in an induction nutrient medium containing dexamethasone or 1,25-dihydroxyvitamin D ₃ , β-glycerophosphate and ascorbic acid-2-phosphate. Several approaches are used to assess the osteogenic differentiation of MSCs [4, 5]. Most often, a histochemical analysis of the cell monolayer is performed to stain calcium deposits in the extracellular matrix using special dyes (either alizarin red S or silver nitrate using the von Kossa method). The presence of mineralization, as a result of otsteogenous differentiation of MSCs, is assessed visually using conventional light microscopy. Less commonly, real-time PCR flow cytofluorimetry or real-time polymerase chain reaction is used to evaluate the expression of surface markers or mRNAs of genes specific for osteoblasts [6, 7]. These approaches are, in fact, qualitative methods that allow only to identify the osteogenic differentiation of MSCs (yes / no) without taking into account its severity and intensity.

To quantify the osteogenic potential of MSCs, calcium levels in supernatants obtained from monolayer and extracellular matrix cell lysates are used. This method requires the stage of extraction of synthesized calcium, for which MSCs are treated with a 0.5 M HCl solution at 4 ° C for 4 hours, then the obtained supernatate is ultracentrifuged, a commercial detection reagent is added and the concentration of calcium (Ca2 +, mmol / μg protein) is measured using photometry at a wavelength of 598 nm. A similar approach to assessing the osteogenic potential of MSCs is based on measuring the activity of alkaline phosphatase (ALP). In this case, the monolayer cell lysate is obtained using a solution of 0.01% NaCl and 0.1% Triton X-100, the resulting supernatant is incubated with a solution of p-nitrophenyl phosphate at 37 ° C, then the enzymatic reaction is stopped by the addition of a 1 M NaOH solution. The activity level of alkaline phosphatase (nM / min / μg protein) is also determined using multichannel photometry at a wavelength of 415 nm and a temperature of 37 ° C using a standard calibration curve of dilutions of r-nitrophenol.

A significant drawback of these methods is the relative complexity of their implementation, associated with the need for 1) an extraction step to obtain a cell lysate, 2) additional reagents for the test itself and the creation of calibration curves, 3) standardization of the obtained results for the protein. These shortcomings not only technically complicate, but also significantly increase the time and cost of laboratory analysis.

The closest solution, chosen as a prototype, is a method for assessing the osteogenic potential of MSCs, based on measuring the optical density of the dye (alizarin red S) used to stain calcium deposits contained in the extracellular matrix of MSCs after their cultivation in an osteogenic induction medium [8]. In accordance with the proposed method, MSCs, after their osteogenic differentiation, are stained standardly with alizarin red S, after which the dye is extracted from the cell monolayer and extracellular matrix with acetic acid, then the resulting supernatant is neutralized with ammonium chloride solution. The intensity of the color reaction of the dye, which is proportional to the amount of calcium deposits formed, is evaluated photometrically at a wavelength of 405 nm. However, to implement this method, a dye extraction step using additional reagents is also necessary, as well as the creation of special conditions (low pH) to increase the sensitivity of the method.

The objective of the invention is to provide a method for the semi-quantitative assessment of the osteogenic potential of mesenchymal stromal cells, which allows to determine the most optimal conditions for the functioning of MSCs depending on the type of tissue source of MSCs, the cell density of MSCs, the multiplicity / duration of passage of MSCs in vitro, the concentration of growth serum factors in the microenvironment etc., in order to increase the efficiency of their subsequent transplantation in the field of traumatology and orthopedics.

The problem is solved in that in the proposed method, the assessment of the osteogenic potential of MSCs is achieved by measuring the optical density of the dye in whole cultures of MSCs in the range of cell concentrations from 625 to 10,000 cells / well and then determining the osteogenic differentiation index in the form of the O / K ratio, where O is the optical density in test samples of MSCs and K, in control samples of MSCs cultured in the same range of cell concentrations without the addition of osteogenic differentiation inducers. The proposed method allows to evaluate the effectiveness of osteogenic differentiation of MSCs in vitro and can be used to analyze the functional activity of MSCs isolated from various tissue sources, as well as to characterize MSCs in the process of their cultivation in vitro or in various pathological conditions. In addition, the proposed method in comparison with the prototype greatly simplifies the technology for assessing the osteogenic potential of MSCs and allows you to reduce material costs and time required for analysis.

The method is as follows.

Bone marrow, adipose or placental tissue are used as starting material for MSCs. Bone marrow is obtained by aspiration from the iliac wing. According to the standard method, 5 to 100 ml of bone marrow are aspirated, the resulting aspirate is placed in a sterile heparin solution at a rate of 50 U / ml and incubated at 37 ° C for 40 minutes. After gravitational separation of the whole bone marrow, the “upper” fraction is collected in a separate vial, consisting of leukoprecipitated cells in the autoplasma. After centrifugation (1500 rpm, 8 min), the cell suspension of the suspension was resuspended in phosphate-buffered saline (PBS). Then, the suspension cells are layered on the density gradient of ficoll-verographin (1.078 g / l) and centrifuged for 20 min at 3000 rpm. The bone marrow mononuclear cells (MNC-km) collected from the interphase are washed three times in PBS and resuspended in α-MEM medium (BioloT, C-Pb). Count the total number of selected MNCs KM and determine their viability by staining with trypan blue.

Lipoaspirate obtained by liposuction is subjected to enzymatic-mechanical dissociation with 0.1% collagenase solution. The tissue of the placenta obtained by urgent physiological delivery is also subjected to mechanical and enzymatic dissociation with a solution of trypsin, EDTA and collagenase. Count the total number of nucleated cells isolated from adipose or placental tissue (YaSK-gt and YaSk-pt), determine their viability by staining with trypan blue.

To obtain an enriched population of MSCs, the selected MNC-km, YAQ-ya or YaAH-pt are cultured with an initial density of 1 × 10 ⁶ cells / cm ² in plastic bottles (Nunc, area 75 cm ² ) in α-MEM medium containing 20% fetal calf serum, at 37 ° C, in an atmosphere of 5% CO ₂ . After 24 hours of incubation, the culture medium is updated and the fraction of non-adherent cells is removed.

In order to quantitatively expand MSCs, the fraction of cells adhering to the plastic is continued to be cultured until a cell monolayer is obtained that occupies 80-90% of the culture vial area (on average for 14 days with the culture medium being replaced every 4-5 days). Then the enriched population of MSCs is obtained using a standard technique for separating cells from a plastic surface in the presence of a 0.25% trypsin solution and a 0.02% EDTA solution, at 37 ° C, for 8-10 minutes, the total number of isolated MSCs and their viability are calculated .

In order to assess the osteogenic potential of MSCs, the obtained cells are placed in the wells of 96-well flat-bottomed plates in the range of 2-fold increasing concentrations from 625 to 10,000 cells / well in α-MEM medium supplemented with 100 μg / ml gentamicin and 20% fetal calf serum (" BioloT ", St. Petersburg.). After 72 h, the culture medium is completely replaced: in the experimental samples, the α-MEM medium is supplemented with the osteogenic differentiation inducers of MSCs (10 mmol / L β-glycerophosphate, 100 nmol / L dexamethasone and 0.2 mmol / L ascorbate diphosphate), whereas in control samples of MSCs continue to be cultured under standard conditions. Replace the appropriate media (standard and induction) is carried out twice a week. After 18 days, the effectiveness of the osteogenic differentiation of MSCs was assessed by the von Kossa method. For this, the experimental and control cultures of MSCs are fixed in 10% formaldehyde solution for 10-20 minutes, then treated with 1% silver nitrate solution for 20 minutes under ultraviolet irradiation, then washed twice with distilled water and kept in 5% sodium thiosulfate solution for removal of non-specific excess silver. Evidence of the osteogenic differentiation of MSCs is calcium production in the form of extracellular matrix deposits, which are stained with silver nitrate in black. The intensity of the color reaction is assessed using a Multiskan Ascent multichannel photometer (Thermo Electron Corp.) at a wavelength of 492 nm. The obtained optical density results (in units of extinction) in the experimental and control samples are expressed as the Osteogenic Differentiation Index (IOD), which is calculated by the formula: IOD = O / K, where O is the optical density in the experimental and K in the control samples.

Examples of specific performance.

Example No. 1. Using the proposed method for assessing the osteogenic potential of MSCs shows that MSCs isolated from adipose tissue differentiate in the osteogenic direction, while the peak production of calcium deposits is recorded in cultures containing 5000-10000 cells / well.

Table 1
Optical density (ext.) And osteogenic differentiation index of MSCs isolated from adipose tissue The number of MSCs / well 625 1250 2500 5000 10,000 MSCs of adipose tissue (n = 6) The control 0.118 0.117 0.102 0,098 0,091 Experience 0.127 0.127 0.162 0.179 0.149 iodine 0.97 1.09 1.01 1.51 1,57 0.91 ± 0.07 1.05 ± 0.06 1.15 ± 0.15 1.68 ± 0.37 1.61 ± 0.14 Note: data are presented as Median and M ± SE

From the data of table 1 it is seen that in the control cultures, regardless of the initial concentration of cells, the optical density is on average 0.100 units. ext., which, obviously, is associated with the formation of a dense monolayer of cells on the surface of the hole during prolonged cultivation. The presence of factors in the culture medium that induce osteogenic differentiation of MSCs is manifested by an increase in optical density in the experimental samples due to the formed deposits of calcium stained in black light. The peak increase in optical density, on average by 51-57%, occurs in cultures with an initial MSC content of 5000-10000 cells / well.

Example No. 2. Using the proposed method for assessing the osteogenic potential of MSCs shows that MSCs isolated from placental tissue are also capable of differentiating in the osteogenic direction under given cultural conditions. At the same time, however, distinctive features of the production of calcium deposits are revealed. From the data of table 2 it is seen that, in contrast to adipose tissue MSCs, a peak in the increase in optical density is recorded at an initial concentration of placental MSCs of 2500 / well, which then gradually decreases in cultures with a higher amount of MSCs (5000 / well), reaching a basal level in the wells, containing 10,000 cells. But even with optimal cellular concentration, the osteogenic potential of placental MSCs is lower than fatty MSCs (IOD 1.39-1.23 vs. 1.51-1.57, respectively).

table 2
Optical density (ext.) And the index of osteogenic differentiation of MSCs isolated from placental tissue The number of MSCs / well 625 1250 2500 5000 10,000 MSCs of the plantent (n = 4 The control 0,101 0.124 0,111 0.113 0.129 Experience 0,076 0.117 0.139 0.150 0,111 iodine 0.74 1.17 1.39 1.23 1,08 0.80 ± 0.18 1.18 ± 0.16 1.38 ± 0.18 1.28 ± 0.9 0.97 ± 0.16 Note: data are presented as Median and M ± SE

Example No. 3. Using the proposed method for assessing the osteogenic potential of MSCs shows that a change in the cultivation conditions or properties of the MSC itself is accompanied by a change in their differentiating osteogenic potential. Table 3 presents the data of a comparative analysis of the intensity of production of calcium deposits of MSCs of adipose tissue, differing in the duration of pre-cultivation.

Table 3
Optical density (ext.) And the index of osteogenic differentiation of adipose tissue MSCs, differing by the duration of preliminary cultivation The number of MSCs / well 625 1250 2500 5000 10,000 MSC (n = 6) after 1 2 passages Experience 0.127 0.127 0.162 0.179 0.149 Iodine 0.97 1.09 1.01 1.51 1,57 0.91 ± 0.07 1.05 ± 0.06 1.15 ± 0.15 1.68 ± 0.37 1.61 ± 0.14 MSC (n = 1) after 9 passages Experience 0,081 0,086 0,085 0,088 0.104 Iodine 1.17 1,11 1,0 1.05 1.17 Note: data are presented as Median and M ± SE

It can be seen that, in contrast to cells that were cultured on average for 20 days before osteogenic differentiation (1-2 passages), MSCs obtained after 9 passages (120 days of cultivation) were resistant to osteogenic signals and did not produce calcium deposits, which, obviously related to the well-known phenomenon of “cell aging”.

From the data of table 4 it is seen that in the conditions of a decrease in the percentage of fetal calf serum (FCS) in the culture medium (from 20 to 10 and 5%), the ability of MSCs to differentiate in the osteogenic direction changes significantly.

Table 4
Index of osteogenic differentiation of MSCs isolated from adipose tissue and placenta under conditions of FCS deprivation in culture medium The number of MSCs / well 1250 2500 5000 10,000 MSCs of adipose tissue (n = 1) FCS 20% 0.78 1,04 1,53 2.18 FCS10% 0.91 1.10 1.74 2,31 FCS 5% 0.91 1.13 2.28 3.20 Placental MSCs (n = 1) FCS 20% 1,50 1,53 1,52 0.52 FCS10% 1,07 1.13 1.10 1.12 FCS 5% 0.90 0.96 1.09 1,04

At the same time, specific features of MSCs of various tissue origin are also found. MSCs isolated from adipose tissue were characterized by an inverse relationship between the level of production of calcium deposits and the content of FCS in the culture medium, i.e. the maximum intensity of their osteogenic differentiation was observed at the lowest 5% concentration of fetal serum. On the contrary, placental MSCs were characterized by a direct dependence of the effectiveness of their osteogenic differentiation on the content of serum factors in the culture medium.

Example No. 4. Using the proposed method for assessing the osteogenic potential of MSCs shows that MSCs isolated from the bone marrow of patients with various diseases differ in their functional activity and ability to differentiate in the osteogenic direction.

Table 5 presents the values of the indices of osteogenic differentiation of MSCs isolated from bone marrow aspirate in patients with spinal trauma (SpTr): 3 patients aged 38-40 years and 2 children aged 4 and 9 years; patients with cirrhosis of the liver (CP, n = 4, 36-48 years old), acute lymphoblastic leukemia (ALL, n = 1, 31 years old) and systemic lupus erythematosus (SLE, n = 1.41 years).

Table 5
Osteogenic differentiation potential of MSCs isolated from the bone marrow of patients with various diseases The number of MSCs / well 625 1250 2500 5000 10,000 SpTr (n = 3, adults) iodine 0.90 0.88 0.94 1.01 1.26 0.77 ± 0.19 0.76 ± 0.22 0.93 ± 0.09 1.18 ± 0.42 1.72 ± 0.56 SpTr (n = 2, children) iodine 1.27 1,64 1.32 1,04 1,08 1.27 ± 0.13 1.64 ± 0.01 1.32 ± 0.15 1.04 ± 0.08 1.08 ± 0.10 CPU (n = 4) iodine 1.14 1.38 1.38 1.27 2.05 1.25 ± 0.15 1.32 ± 0.19 1.46 ± 0.13 1.42 ± 0.28 1.77 ± 0.38 ALL (n = 1) iodine 0.96 1,08 0.97 0.98 0.90 SLE (n = 1) iodine 1,1 0.97 0.98 1,04 1.18 Note: data are presented as Median and M ± SE

Thus, the proposed method for measuring at a wavelength of 492 nm the optical density of whole MSC cultures in the range of cell concentrations from 625 to 10,000 cells / well, after culturing them in an osteogenic induction medium and staining calcium deposits with silver nitrate, allows a semi-quantitative assessment of the effectiveness of osteogenic differentiation of MSCs in vitro. The proposed method can be used to increase the efficiency of MSC transplantation in the field of traumatology and orthopedics, since it allows you to determine the most optimal conditions for the functioning of MSCs depending on the type of tissue source of MSCs, the cell density of MSCs, the multiplicity / duration of passage of MSCs in vitro, the concentration of growth serum factors in the microenvironment , the nature of concomitant pathology. An additional advantage of the proposed method is the absence of the need for the stage of extraction of the dye from the monolayer of cells and the extracellular matrix, which allows, without reducing the quality of the analysis, to simplify the technology of its implementation by reducing material and time costs.

Literature

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Claims (4)

1. A method for assessing the osteogenic potential of mesenchymal stromal cells, including photometric measurement of the optical density of the dye used to stain calcium deposits contained in the extracellular matrix of MSCs after their cultivation in an osteogenic induction medium, characterized in that the optical density of the dye is measured in whole MSC cultures in a given a range of 2-fold increasing cell concentrations, silver nitrate is used as a dye, while the intensity of the color reaction and evaluated on a multi-channel photometer at a wavelength of 492 nm, and then the osteogenic differentiation index is calculated as the O / K ratio, where O is the optical density in the experimental samples of MSCs and K is in the control samples of MSCs cultured in the same range of cell concentrations without adding inducers of osteogenic differentiation. 2. The method according to claim 1, characterized in that cells isolated from various tissue sources, for example, from bone marrow, adipose, placental tissue, can be used as MSC cultures. 3. The method according to claim 1, characterized in that as cells of MSCs, cells isolated from the bone marrow of patients with various pathological conditions can be used. 4. The method according to claim 1, characterized in that as the cultures of MSCs, cells obtained under various in vitro cultivation conditions can be used.