RU2484472C1 - Method for morphofunctional assessment of cell component of biografts - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: for the purpose of dynamic studies, sterile biografts are placed into cultural containers in a laminar flow hood, and coated with a cell suspension in a nutrient medium; sterile vital fluorochrome in a buffer solution containing trypaflavine and rhodamine C is introduced. The cultural cell containers are incubated; the cell culture on a biograft is analysed daily. An average light intensity of a visual field (ALIVF) is derived from a calibration curve. The number of attached cells (NACs) is specified per 1 unit of a biograft area, th/cm². The total number of attached cells (TNACs) on the biograft is specified, thousands. A proliferation index (PI) is calculated. An average luminous intensity of 1 cell on the biograft is calculated, foot-candles and expressed in points, where 1 foot-candle is 1 point that characterises the cell membrane integrity (CMI). The killed cells (KCs) are counted, %. The transplantation suitability of the cellular component of the biograft is assessed.

EFFECT: invention provides the objective morphofunctional dynamic assessment of the cell component of the biograft, developed or used for transplantation, due to taking into account the data obtained through the study of the vital stained cells in the fluorescence microscope.

1 cl, 4 ex, 1 dwg, 6 tbl

Description

The invention relates to medicine and can be used to assess the quality of biografts used in transplantology, in reconstructive and plastic surgery, traumatology, combustiology, in the treatment of patients with emergency conditions.

The provision of high-tech medical care, defined by the government as one of the main tasks of the development of healthcare, is impossible without the widespread introduction of transplantation of organs, tissues and cells into clinical practice. Currently, cell and tissue transplantology is considered as one of the promising directions in the treatment of a wide range of pathologies. The development of cellular technologies in the field of regenerative medicine leads to the creation of new composite materials compatible with cells, the formation of three-dimensional tissue-like structures intended for transplantation [On the development of cellular technologies in the Russian Federation. MOH CP RF №325 from 07.25.2003. The use of cell therapy in clinical practice. Actual issues of tissue and cell transplantology: Collection of theses of the fourth All-Russian symposium with international participation / Edited by Acad. Mironova S.P. - St. Petersburg: Publishing House "Man and His Health", 2010. - 334 p.]. Fibroblasts, bone marrow cells, endotheliocytes, hepatocytes are used to improve the regeneration of bone and cartilage tissue, restore the skin, blood vessels and liver in surgery, combustiology, traumatology [Grudyanov AI, Erokhin AI, Mironova L., Konyushko O.I. Laboratory study of the activity of fibroblasts in combination with various types of planting materials in vitro. Cytology 2001; 43: 9: 854. Grudyanov A.I., Erokhin A.I. "The method of vestibuloplasty", Patent for the invention of the Russian Federation No. 2162663 of 02/10/2001; Shurygina O.V. The use of cell culture of allogeneic fibroblasts in the complex treatment of alveolitis. diss. Cand. honey. sciences. M., 2006. S. 29; A.S. Ermolov, S.V. Smirnov, V. B. Khvatov et al. "Method for the treatment of burn wounds", patent for the invention of the Russian Federation No. 2373944 from 11.27.09; Kolokoltsova T.D., Nechaeva E.A., Yurchenko N.D. et al. "HUMAN DIPLOID CELL STRAIN FOR SUBSTITUTE THERAPY (OPTIONS)", patent for invention of the Russian Federation No. 2285040 C2 (51) dated 10.10.2006]. Subsequently, the development of new biologically active materials based on human and animal tissues, the creation of composite biografts with various carriers, including living cells, began. This is reflected in the collection of abstracts of the Fourth All-Russian Symposium with international participation “Actual Issues of Tissue and Cell Transplantology”, St. Petersburg, 2010, experts suggest for clinical use: a cell-tissue transplant based on demineralized spongy bone material containing multipotent mesenchymal stromal cells from bone marrow (Volova L.T. et al., P. 42); tissue engineering equivalent of bone (Deev R.V. et al., p. 62); combined cell-tissue transplant for plastic surgery of defects in articular hyaline cartilage (Kotelnikov G.P. et al., p. 81); the use of multipotent mesenchymal stromal cells (mm sc) on demineralized bone-cartilage matrices for the restoration of damaged articular surfaces (Grigoryan A.S. et al., p. 161); biodegradable polymer carriers for the cultivation and transplantation of human skin cells, (Shved, Yu.A. et al., p.202); multipotent mesenchymal stromal cells as part of hydrogels, microspheres, and wide-pore cryogels based on alginate (Petrenko A.Yu. et al., p. 177); composite materials based on polymer matrices with integrated stem cells and nanocomponents (Smirnova N.V. et al., p. 187).

Thus, the manufacture of biografts is associated with the widespread use of different types of cultured human cells. This inevitably raises the problem of objective control of the interaction of cell cultures in the composition of the proposed biotransplant, primarily with the determination of the number and quality of cells.

There is a method for estimating the number of cell cultures in suspension using a counting chamber (M. Benyumovich, “Counting chamber with grids”, RF patent No. 212630, IPC АВВ 10/00, 1999.02). However, this method cannot be used, because its use requires the separation of cells from the biotransplant, which is technically impossible when creating a monolayer of cells; in addition, this method does not allow for a full-fledged qualitative analysis of cells. Note that a comprehensive assessment is required to characterize any cell culture. This integral assessment of the culture consists of indicators such as the ratio of living and dead cells, the dynamics of cell population growth, the rate of monolayer formation (Gerasimov I.G., Popandopulo A.G. "Assessment of cell viability by their morphometric parameters on the example of cultured fibroblasts" / / Cytology. 2007.49 (3) S.204-209).

Prototype: Kvitko O.V .; Nikolaevich L.N. A method of analyzing a human fibroblast population (RF patent No. 20177818, 08/15/1994). Human fibroblasts obtained from skin and muscle tissue of 7-8-week-old human embryos are cultured in Eagle medium containing 10% cattle serum. In the logarithmic growth phase, cells are trypsinized with a 0.02% Versen solution and 0.25% trypsin solution at 4 ° C (2 min), followed by incubation in an incubator at 37 ° C (up to 10 min). On Petri dishes (with a diameter of 35 mm) seeded cells at a concentration of 100-500 cells / mm ² . Cells are incubated under conditions of saturation humidity at 37 ° C with 5% CO ₂ for 20-24 hours. To determine the concentration of cells in the culture, the method of counting cells in the Goryaev chamber is used.

The normal human fibroblast population includes two subpopulations of cells. Some fibroblasts have a high proliferative potential and, when cloned, form multicellular colonies (type I), while others have a low mitotic potential and under clonal conditions produce small-cell, loose colonies (type II). Morphologically, the former are spindle-shaped cells, and the latter are sail-like and pleomorphic cells. Type I fibroblasts differentiate into type II fibroblasts. With the age of the donor and with the aging of fibroblast cultures, cells with reduced proliferative potential (sail-like and pleomorphic) accumulate. For cytomorphological analysis, the cells are fixed and treated with 2% orcein in 45% acetic acid. A cytomorphological analysis of the structure of the fibroblast population by the ratio of spindle-shaped, sail-shaped and pleiomorphic cells is carried out using a microscope (MBI-9), using a special grid of 8 × 8 mm in size, with a division price of the side of the square 0.5 × 0.5 mm, which is installed in the eyepiece a microscope. Analyze 10 fields of view, selected involuntarily in the clockwise direction. Five replicates (Petri dishes) are placed on each point. It was shown that both in stationary and radiation-induced aging of the culture, a gradual, especially pronounced in the last term (20 days), decrease in the proportion of fusiform cells occurs. Thus, the results obtained indicate that the accumulation of inactive cells (type II) is the cause of the natural and radiation-induced aging of cell populations.

The disadvantages of this method are: the need for trypsinization of cells to count them in the Goryaev chamber and the errors associated with this; the need for cell fixation for cytomorphological analysis; unsuitability of cytomorphological criteria for fibroblast activity for work with other cell cultures; the inability to determine the ratio of living and dead cells in culture; the impossibility of dynamic monitoring of cell culture in the composition of biografts.

Thus, it is required to develop an adequate assessment method in the dynamics of the interaction of the cellular component with the biotransplant used or being developed for transplantation in clinical practice. It is justified to use vital staining methods to assess the interaction of cell cultures with a biograft. This will allow research on non-fixed objects, i.e. living cells, continuously evaluate the dynamics of their growth and division, changes in the properties of cells grown on various substrates. In this case, the morphological evaluation of substrate-linked cells is carried out immediately before clinical use, without detaching the cell culture from the biotransplant.

There are a number of vital dyes that, under the influence of exciting light, cause fluorescence of the cytoplasm of cells without visible disturbances in their vital functions [Romeys B. Microscopic technique. - M .: Publishing house of foreign literature, 1954. - S.171-172]. Of these dyes, we selected tripaflavin and rhodamine C. Tripaflavin has a high penetrating ability and in small concentrations does not cause disturbances in the genetic apparatus of cells. Rhodamine C intensively stains the membrane structures of cells and also does not interact with the cell genome. In addition, both of these dyes have the ability for a long time (up to 7 days) to maintain their fluorescent color. Thus, there are good reasons for using a complex fluorescent dye that would have high penetrating power, low toxicity, and long-lasting glow.

Achievable technical result is an objective dynamic morphofunctional assessment of the cellular component of a biograft developed or used for transplantation, by taking into account data obtained by studying vitally stained cells in a fluorescence microscope.

The manufacture of dye. The preparation of the dye, including fluorochromes, tripaflavin and rhodamine C, consists of two stages. At the first stage, concentrated solutions of tripaflavin (solution A) and rhodamine C (Solution B) are prepared in 0.15 M phosphate buffer: Solution A - tripaflavin is diluted with 0.15 M phosphate buffer in a ratio of 1: 2000-1: 5000. It is necessary to achieve complete dissolution of tripaflavin crystals. Solution B - rhodamine C is diluted with 0.15 M phosphate buffer in a ratio of 1: 1500-3000. Then both dyes are mixed in a ratio of 1: 1 and diluted 10 times with 0.9% NaCl solution. This is a working solution of vital dye. To stain 1 cm ^{2 of the} biograft with cells, 2-5 μl of the dye working solution is required.

Vitally stained cell culture in the biograft is evaluated using a fluorescence microscope (lens × 4, numerical aperture 1.25) in a semi-automatic mode. To obtain a fluorescence image, use a green filter (λ excitation 510-560 nm, λ emission - from 590 nm). Using a camera at an exposure of 1-4 seconds, digital images of cells are obtained and transferred to a computer. The unit of analysis is 1 field of view of the microscope, which is 5 mm ^{2 of the} area of cell or matrix culture. For morphometric studies of cells, assessing the brightness of the luminescence of 1 field of view using the program Adobe Photoshop. An analysis of the nature and intensity of luminescence of intravitally stained culture cells on a biograft can be observed for 3-4 days. For a longer observation, 1 μl of culture medium is supplemented with 10 μl of a sterile working solution of tripaflavin and rhodamine C every 3-4 days.

In experiments, it was found that the culture cells stained with vital dye tripaflavin and rhodamine C retain their proliferative properties (table 1) and viability (table 2). The proliferation index, the time of monolayer formation, the number of living and dead cells in the culture and on bioimplants in the group without and after staining with vital fluorochrome dyes were identical. The results of these studies substantiate the adequacy of the proposed morphofunctional assessment of various cell cultures using intravital staining with tripaflavin and rhodamine C.

Table 1 Comparison of the proliferative properties of various cell cultures without and during vital staining of cells (analysis for 7 days of cell culture) Type of culture The presence of vital dye in cell culture The number of cells in the culture (thousand) per 1 Petri dish Proliferation index The time of formation of the monolayer (days) M22 fibroblasts absent 156 2.86 5 is present 150 2.75 5 Tissue donor skin fibroblasts absent 120 1.9 7 is present 125 1.7 7 Human bone marrow mesenchymal stem cells absent 35 2.10 19 is present 28 1.94 twenty Human Chondroblasts absent 180 2.95 four is present 190 2.90 four Rat skin fibroblasts absent 140 2,3 7 is present 150 2.2 7

table 2 Comparison of the viability of various cell cultures without and during vital staining of cells Type of culture The presence of vital dye in the culture The content of dead cells in the culture at different periods of passage, (in%) 1 day 3 days 7 days Human fibroblasts of the M22 line absent 0.1 0.1 0.5 is present 0.2 0.3 0.5 Tissue donor skin fibroblasts absent 1,1 2.0 5,0 is present 1,5 3.0 6.0 Human bone marrow mesenchymal stem cells absent 0.2 0.4 0.7 is present 0.3 0.6 1,0 Human Chondroblasts absent 0.1 0.1 0.2 is present 0.1 0.2 0.5 Rat skin fibroblasts absent 0.5 0.7 2.0 is present 0.5 1,0 2,5

Parameters for assessing the cell population in bioimplants

1. The number of attached cells per unit area of the biograft - CPC (thousand / cm ² )

The number of attached cells (CPC) per unit area is estimated by the intensity of the luminescence (brightness) in a standard (5 mm ² ) field of view of the microscope with cells vitally stained with tripaflavin and rhodamine C. Between the intensity of the luminescence of the standard field of view (in footcandles) and the number cells in the standard field of view of a fluorescence microscope, a close correlation was found, confirmed by the equation y = 1.1861x + 0.89 (Fig. 1, where the OX axis is the number of cells per unit area, thousand / cm ² , the OA axis is the average brightness of one field view foot candela). To calculate the CCP per unit area, a calibration curve is constructed that reflects the dependence of the number of cells per unit area of the bottom of the culture vial, Petri dish, or biotransplant on the intensity of the glow of the microscope field of view (5 mm ² ).

Construction of a calibration curve. Using the Fuchs-Rosenthal counting chamber, the number of cells in 1 ml of the analyzed cell suspension is determined. In several Petri dishes (from 4 to 7) with a diameter of 4 cm containing the culture medium, a suspension with a known number of cells, for example 50, 75, 100, 125, 150 and 200 thousand, is added. After this, Petri dishes were placed in a thermostat at 37 ° C for 24 hours to attach viable cells. Then the Petri dishes are removed from the thermostat, the culture medium is removed from them, the cell culture is stained with tripaflavin and rhodamine C, at the rate of 5-10 μl per 1 cm ² of surface area. On the outside of the bottom of the Petri dish using a marker, enter a square divided into 9 identical squares, the total area of which is at least 70% of the area of the bottom of the cup. The cup is placed on a stage of a fluorescence microscope. Register (lens x4) image on 3 fields of view of the microscope in each of 9 squares.

Next, the image is transferred to a computer and using the Adobe Photoshop program, the brightness of each field of view (in foot-candels) is estimated, the average brightness of the luminescence of 1 standard (5 mm ² ) field of view of the microscope is calculated by the formula and expressed in foot-candes. Average brightness of the visual field (SPL) = total brightness of the glow of the examined visual fields: the number of examined visual fields

The number of attached cells per unit area for each dish is determined (in the above example, 4.2, 6.3, 8.4, 10.5, 12.6 and 16.8 thousand cells per cm ² , respectively) and based on these data, calibration curves are constructed for different types of cells, showing the relationship between CPC and SYAP.

For example. In the study of the culture of human fibroblast cells of the M22 line cultured in a Petri dish, in the study of 27 standard fields of vision, the SPE was 10 foot-candela. This value on the calibration curve of the CPC corresponds to 7.7 thousand cells / cm ² or according to the formula y = 1.1861x + 0.89, y = 10 ft-candela, CPC = 7681 c / cm ² .

For example. In the study of chondroblasts planted on the bone matrix, in the study of 27 standard visual fields, the SPE was 5 foot-candelas. To this value on the calibration curve of the CPC corresponds to 3.5 thousand / cm ^{2 of} bone matrix or according to the formula y = 1.1861x + 0.89, y = 5 foot candela, CPC = 3465 cells / cm ²

2. The total number of attached cells OKPK (in thousands)

OCCP reflects the content of cells on the studied surface of the biograft, three-dimensional tissue-like structures intended for transplantation.

OKPK is determined by the formula: OKPK = KPK × Sp, where KPK is the number of cells per 1 cm ² (in thousand / cm ² ), Sp. - the area of the investigated surface of the culture tank or biotransplant (in cm ² ).

For example. The bottom area of the Petri dish is 12 cm ² , the CCP is 17 000 / cm ² .

OKPK = 17000 cells / cm ² × 12 cm ² = 204000 attached cells in a Petri dish. OKPK = 204000 cells.

For example. The area of the collagen-based biotransplant used to treat burns is 144 cm ² , the fibroblast CPC is 6000 / cm ² . OKPK = 6000 fibroblasts / cm ² × 144 cm ² = 864000 attached fibroblasts on a biograft.

3. The proliferation index (PI) of cell culture on biotransplant

This indicator reflects the growth activity of the cell population in culture, i.e. how many times the total number of cells (OKPK) on a given day of passage exceeds the number of cells on the 1st day of passage. PI depends on the type of cells and the timing of their cultivation.

Based on the data of the CPPC obtained during long-term observation of cell culture (for example, in dynamics over 7 days), it is possible to calculate the proliferation index of cell culture in a Petri dish or on a biotransplant.

IP = OKPK analyzed: OKPK source.

For example, on day 1 of passage of OKPK ₁ = 50,000, on day _{4 of} passage of OKPK ₄ - 110,000, on day _{7 of} OKPK ₇ - 140,000 cells. The proliferation index of cell culture on the 4th day of passage is equal to OKPK ₄ : OKPK ₁ = 110000: 50000 = PI ₄ = 2.2. Accordingly, the proliferation index on the 7th day of passage is equal to OKPK ₇ : OKPK ₁ = 140,000: 50,000 = PI ₇ = 2.8.

4. The integrity of cell membranes - CCM (in points) is estimated by the intensity of the glow of the cell

This parameter reflects the quality of cell membranes, which is associated with the viability of the cells that are part of the biograft. CCM is estimated by the intensity of the glow of cultured cells. To do this, in a fluorescence microscope under a × 20 lens, 100-150 cells vitally stained with fluorochromes trypavlavin and rhodamine C cells are analyzed on the test surface. The resulting digital image is transferred to a computer. Using the program Adobe Photoshop evaluate the intensity of the glow (brightness) of each cell. Determine the average brightness of 1 cell in foot candelas. CCM is evaluated in points, where 1 point corresponds to 1 foot candela.

For example, in the study of 120 cultured cells in a biograft, the intensity of the brightness of their luminescence ranged from 29 to 38 foot-candelas, averaging 35 foot-candelas per 1 cell or 35 points. Therefore, the CCM of the analyzed cell population is 35 points.

In different cell cultures, CCMs vary from 25 to 50 points depending on the type and size of cells. Dead cells have very low values of CCM - 5-15 points. A classification of the integrity of the membranes in the population of cell culture is proposed (table 3).

Table 3 Cell membrane integrity (in points) Cell Characterization in Culture 5-15 Dead cells 16-27 Reduced viability cells 28-42 Normal viability cells ≥42 Overactive cells

5. The content of dead MK cells (in%)

This parameter is estimated by the formula: MK = the number of cells with CCM less than 15 points is divided by the number of all examined cells and expressed as a percentage.

For example, out of 100 cells examined, 3 cells had MCC = less than 15 points. MK = 3: 100 = 3.0%; of 120 cells examined, 25 cells had MCC = less than 15 points. MK = 25: 120 = 20.8%.

For a better understanding of the proposed method, we give the following examples.

Example 1. Investigated the growth dynamics of the culture of fibroblasts of human skin for 7 days in a Petri dish with an area of 12 cm ² . Fibroblasts before seeding under sterile conditions were stained vitally at the rate of 10 μl of dye per 1 ml of cell suspension. The initial total number of introduced cells was 50,000. The cells were attached at the bottom of the Petri dish 1 day after plating. An analysis of the fibroblast culture was performed on days 1, 2, 3, 4, 5, and 7 after seeding. The average brightness of the visual field (SPL), the number of attached cells per unit area (CPC), the total number of attached cells (CPC), the proliferation index (PI) of the cell culture, the integrity of the cell membranes (CCM), and the content of dead cells (MC) were evaluated.

The parameters for evaluating the cell population in culture were as follows:

on the 1st day - SPL = 4.9 ft-candela, CPC = 3381 / cm ²

on the 2nd day - SPL = 6.2 ft-candela, CPC = 4652 / cm ²

on the 3rd day - SPL = 12.1 ft candela, CPC = 9452 / cm ²

on the 4th day - SPL = 15.3 ft-candela, CPC = 12150 / cm ²

on the 5th day - SPL = 15.5 ft candela, KPK = 12318 / cm ²

on the 7th day - SPL = 15.6 ft candela, KPK = 12 401 / cm ²

on the 1st day - OKPK = 40572, -

on the 2nd day - OKPK = 55824, IP ₂ = 1.37

on the 3rd day - OKPK = 113424, IP ₃ = 2.79

on the 4th day - OKPK = 145800, IP ₄ = 3.59

on the 5th day - OKPK = 147816, IP ₅ = 3.64

on the 7th day - OKPK = 148812, IP ₇ = 3.67

on the 1st day - CCM - 31 points, MK - 0.5%

on the 2nd day - CCM - 31 points, MK - 0.5%

on the 3rd day - CCM - 31 points, MK - 0.5%

on the 4th day - CCM - 31 points, MK - 0.7%

on the 5th day - CCM - 30 points, MK - 2.0%

on the 7th day - CCM - 30 points, MK - 3.0%

Conclusion: the analyzed fibroblast cell culture has normal growth properties and is recommended for clinical use.

Example 2. For clinical use, various types of biografts were prepared — based on collagen, a polymer of hyaluronic acid, and a human dermis. M22 line fibroblasts were sown on these biografts (0.5 million cells each) and the cellular component of biografts was monitored for 3 days (Table 4).

Table 4 Assessment of cell culture in biografts after 3 days of cultivation Rating Parameter Collagen-based biograft with fibroblasts (S = 144 cm ² ) Collagen-based biograft with fibroblasts and fibroblast growth factor (S = 144 cm ² ) A human dermis biograft with fibroblasts (S = 144 cm ² ) Hyaluronic acid polymer biotransplant with fibroblasts
(S = 144 cm ² ) SYAP (foot candel) 11.8 21.0 7.2 0.5 PDA (cells / cm ² ) 9198 16954 5320 Cells do not attach OKPK (cl) 1324542 2441480 766080 Cells do not attach IP 1.9 3,5 1.4 - CCM (in points) 35 35 thirty MK (in%) 2.0 5,0 7.0 - Conclusion Cell culture is normal. Biotransplant recommended for clinical use Cell culture is normal. Biotransplant recommended for clinical use Cell culture contains a reduced number of cells. Biotransplant is not recommended for clinical use. Cells did not adhere to the carrier. Biotransplant does not contain a cellular component

Example 3. Investigated the effect of EHF irradiation on a fibroblast culture line M22, planted on a biograft based on collagen with an area of 144 cm ² . Evaluation of the culture in the biotransplant was performed before EHF irradiation and 3 days after EHF irradiation (table 5).

Table 5 Rating Parameter Collagen-based biograft with fibroblasts before EHF irradiation Collagen-based biograft with fibroblasts (3 days after EHF irradiation) SYAP (foot candel) 11.8 7.2 PDA (cells / cm ² ) 9198 3694 OKPK (cl) 1324542 531995 IP 1.9 1.4 CCM (in points) thirty 19 MK (in%) 2 33 Conclusion Cell culture is normal. Biotransplant recommended for clinical use A noticeable decrease in the number of cells and a decrease in their viability in culture is observed. Biotransplant is not recommended for clinical use.

Example 4. Investigated the growth dynamics of mesenchymal stem cells (MSCs) of the bone marrow of an organ donor on a biograft based on demineralized human bone with an area of 12 cm ² . Evaluation of the culture in the biotransplant was performed 1 day and 4 days after cultivation (table 6).

Table 6. Rating Parameter Demineralized bone biograft 1 day after seeding of MSCs Demineralized bone biograft 4 days after seeding of MSCs SYAP (foot candel) 7 7.5 PDA (cells / cm ² ) 5932 6336 OKPK (cl) 71184 76032 IP - 1,07 CCM (in points) thirty 22 MK (in%) 2 fourteen Conclusion Continue cell culture on biograft MSCs on biotransplant have reduced viability. The number of dead cells is increasing. Continue culturing MSCs

Summarizing the above, the proposed method is as follows:

For dynamic research in a laminar cabinet, sterile biotransplants are placed in the culture containers, onto which a suspension of cells is added in a nutrient medium, a sterile vital fluorochrome dye is added on a buffer solution containing trypavlavin and rhodamine C in a ratio of 1: 1, moreover, for staining 1 cm ² of biotransplant cells 5-10 μl of the dye working solution are required,

- culture containers with cells are incubated in conditions of 5% CO ₂ at 37 ° C,

- daily analyze the cell culture on the biotransplant, for which the culture containers are moved to the stage of the fluorescence microscope - x4 lens, aperture 0.6, the excitation wavelength of 510-560 nm, the emission wavelength - from 590 nm, and measured in foot channels on biotransplant with cells, the brightness of the luminescence 27 standard fields of view of the microscope,

- calculate the average brightness of the glow of the field of view (SPL) on the calibration curve,

- determine the number of attached cells (CPC) per 1 unit of biotransplant area, in thousand / cm ² ,

- calculate the total number of attached cells (OKPK) on the biotransplant, in thousands,

- calculate the proliferation index (IP), for which it is determined how many times the CPC on a given day of passage exceeds the number of cells on the 1st day of passage,

- under the lens × 20 analyze the image of 100-150 cells on a biograft,

- calculate the average value of the intensity of the luminescence of 1 cell on the biograft in foot candelas and expressed in points, with 1 foot candel = 1 point, which characterizes the integrity of cell membranes (CCM),

- determine the percentage of cells with low brightness, which reflects the content of dead cells (MK), in%,

- assess the suitability of the cellular component of the biograft for transplantation, which normally contains 14-20 thousand cells per cm ² , of which no more than 5% are dead, CMC 28-42 points, PI on the 3rd and 7th day of passage varies from 1.8 up to 3.2 depending on the type of cells.

Claims (1)

The method of morphofunctional assessment of the cellular component of biografts, which consists in the fact that sterile biotransplants are placed in culture containers for dynamic research in a laminar cabinet, onto which a suspension of cells is added in a nutrient medium, sterile vital fluorochrome dye is added on a buffer solution containing trypaflavin and rhodamine C in the ratio 1: 1, and 1 for dyeing ^cm2 biotransplantata cells requires 5-10 l of working dye solution, and then the culture vessel with a cell incubus ruyut under 5% CO ₂ at 37 ° C, then assayed daily for cell culture biotransplantate, for which the culture vessel is moved on a fluorescence microscope stage - 4 × lens, aperture 0.6, wavelength 510-560 nm excitation wavelength emission is from 590 nm, and the brightness of the luminosity of 27 standard fields of view of the microscope is measured in the footcandles on a biograft with cells, the average brightness of the luminosity of the field of view (CPS) is calculated from the calibration curve, the number of attached cells (CPC) per 1 unit of bio area is determined transplant, thousand / cm ² , then calculate the total number of attached cells (OCPC) on the biotransplant, thousand, calculate the proliferation index (PI), which determines how many times the OCC on the day of passage exceeds the number of cells on day 1 passivation, then under the × 20 lens, the image of 100-150 cells on the biotransplant is analyzed, the average luminous intensity of 1 cell on the biotransplant is calculated in foot candeals and expressed in points, with 1 foot candela = 1 point, which characterizes the integrity of cell membranes (C KM), determine the percentage of cells with low luminosity, which reflects the content of dead cells (MK),%, assess the suitability of the cell component of the biograft for transplantation, which normally contains 14-20 thousand cells per cm ² , of which not more than 5% dead, CCM 28-42 points, PI on the 3rd and 7th day of passage varies from 1.8 to 3.2, depending on the type of cell.

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