RU2537141C1 - Method for assessment of marrow cell fissility - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: cells are stored in a standard stabiliser at room temperature; a first sample is taken from the suspension, and 3-3.5 hours after the suspension storage, a second sample is taken; the samples are exposed to a fluorescent potential-sensitive viral probe of iodide 2-(n-dimethylaminostyryl)-1-methylpyridinium; a probe fluorescence strength is measured in the cells; a cell portion having the probe fluorescence strength of more than 100 standard units is calculated in each probe; if observing the cell portion having the probe fluorescence strength of more than 100 standard units 1.5 times higher than in the second sample as compared to the first sample, the marrow bone fissility is stated. The method enables assessing the spontaneous marrow bone fissility caused by the effect of proper growth factors, and eliminated the reasons causing cell fission and differentiation.

EFFECT: method is technically simple and enables obtaining the results 3-3,5 hours later.

3 tbl, 3 ex

Description

The invention relates to medicine, in particular clinical biochemistry, cytology, pathomorphology, and can be used to determine the ability of bone marrow cells (BMC) to divide.

There is a method of determining the colony forming ability of hematopoietic bone marrow stem cells to assess their viability, based on cultivation on various semi-viscous and semi-solid nutrient media (methyl cellulose, collagen, agar) with the mandatory addition of exogenous growth factors [C. Nissen-Druey, A. Tichelli, S. Meyer-Monard. Human Hematopoietic Colonies in Health and Disease. Acta Haemotologica (ISSN 0001-5792), V.113, No. 1, 2005]. Cultural media are designed to maintain an optimal mode of life of isolated cells, fragments of tissues and organs in artificially created systems. However, most often used as nutrient media agar and methylcellulose affect the proliferation and differentiation of CMC.

A known limiting dilution (LDA) method is known [Takaue Y., Reading CL, Roome AJ, Dicke KA, Tindle S., Chandran, M., Devaraj B. Limiting-Dilution Analysis of the Effects of Colony-Stimulating Factors, Phytohemagglutinin, and Hydrocortisone on Hematopoietic Progenitor Cell Growth. Blood, V.70, No. 5, 1987, P. 1611-1618], based on the cultivation of hematopoietic progenitor cells in a liquid medium and eliminating the effect of agar and methyl cellulose on colony forming ability. The use of the LDA method allowed us to study the effect of phytohemagglutenin, hydrocortisone and various cytokines on hematopoietic progenitor cells.

To study the proliferative activity of cells, a method using oxyurea is used, which inhibits DNA synthesis through action on ribonucleotide reductase. Cells are isolated, washed and incubated with oxyurea, washed again and transferred to a nutrient medium, incubated for 7-9 days (in the control without oxyurea) [Goldberg ED, Dygay A.M., Shakhov V.P. Methods of tissue culture in hematology. Tomsk, 1992, under the editorship of doctor of medical sciences Novitsky, 272 p.].

Common disadvantages of the above methods are:

- the influence of the growth medium on cell division and differentiation;

- the addition of exogenous colony-stimulating factors masks the effect of their own growth factors and does not allow us to assess the spontaneous ability of cells to divide;

- methodological complexity and duration (results are obtained after 7-14 days).

The technical result of the claimed invention is to determine the spontaneous ability of KKM to divide, due to the effect of their own growth factors, as well as simplifying the method, reducing the time to obtain results.

The specified technical result is achieved in a method for determining the ability of bone marrow cells to divide, in which the cells are stored in a standard stabilizer solution at room temperature, the first initial sample is taken from the suspension, and after 3-3.5 hours of storage of the suspension, the second sample is taken, the samples are treated with a fluorescent potential-sensitive with a vital probe iodide 2- (p-dimethylaminostyryl) -1-methylpyridinium (2-Di-1-ASP), measure the fluorescence intensity of the probe in the cells, calculate the proportion of cells with fluorescence intensity entsii over 100 conv in each sample, and with an increase in the proportion of cells with fluorescence intensity of more than 100 conventional units more than 1.5 times in the second sample compared with the first, they judge the ability of bone marrow cells to divide.

The CMC population is extremely heterogeneous and contains a large number of blood cells and progenitor cells of varying degrees of maturity. [Vorobiev A.I., Drize N.I., Chertkov I.L. Blood formation scheme. Prob. Hematology, 1995, v. 1, No. 1, pp. 7-14]. Among the progenitor cells, stem and progenitor cells are distinguished, which give rise to mature blood cells - colony forming units (CFU). Bone marrow stromal cells produce stem cell factor (FCS), which stimulates the proliferation and differentiation of hematopoietic cells.

Previously, the authors found that the fluorescent potential-sensitive vital probe of the cationic type 2-Di-1-ASP penetrates into living CMCs and fluoresces in them, and its fluorescence is sensitive to changes in the energy potential of CMCs [RU No. 2488826, publ. 07/27/2013, bull. No. 21].

When developing the inventive method, samples of a suspension of native CMC in a standard solution of a stabilizer of 20 donors were studied, which were stored at room temperature (approximately 25 ° C). Initially, the proportion of living cells in the samples was 80-92% (trypan blue test [Phelan M.C. Basic techniques for mammalian cell tissue culture. Current Protocols in Cell Biology. 0: 1.1.1-1.1.10. 1998]).

As a standard solution of the stabilizer was used the drug company "Terumo Corporation, Tokyo, Japan", containing in 100 ml:

sodium citrate 2.63 g lemon acid 0.327 g sodium dihydrogen phosphate 0.251 g dextrose anhydrous 2.9 g adenine 0.0275 g distilled water rest

ККМ. В каждом препарате определяли флуоресценцию массива 80-100 клеток. Все исследованные клетки в каждом препарате разделяли на 4 группы по величине интенсивности флуоресценции: I - от 10 до 30 усл.ед.; II - от 30 до 70 усл.ед.; III - от 70 до 100 усл.ед.; IV - от 100 усл.ед. и рассчитывали долю клеток в каждой группе. Количество ядросодержащих клеток определяли по известному методу [Клиническая лабораторная аналитика, том 2. Частные аналитические технологии в клинической лаборатории. Под общей редакцией Меньшикова В.В., М., «Лабинформ», РАМЛД, 1999.], результаты выражали в процентах по отношению к количеству клеток в исходной пробе (NC=C/C_исх.×100).Cell samples were taken from the samples initially (0 hours) and after 1 hour, 2 hours, 3 hours, 5 hours, 24 hours, 48 hours, 72 hours of storage. Samples were incubated with 2-Di-1-ASP at 37 ° C, cell preparations were prepared, examined using a luminescent microscope, and the average fluorescence intensity was determined $$\left(\tilde{F}\right)$$

KKM. In each preparation, the fluorescence of an array of 80-100 cells was determined. All studied cells in each preparation were divided into 4 groups according to the magnitude of the fluorescence intensity: I - from 10 to 30 conventional units; II - from 30 to 70 conventional units; III - from 70 to 100 conventional units; IV - from 100 conventional units and calculated the proportion of cells in each group. The number of nucleated cells was determined by the known method [Clinical laboratory analytics, volume 2. Private analytical technology in a clinical laboratory. Under the general editorship of Menshikov VV, M., "Labinform", RAMLD, 1999.], the results were expressed as a percentage in relation to the number of cells in the initial sample (NC = C / C _out. × 100).

Statistical processing of these experiments was carried out according to t student criterion [Bailey I.N. Statistical methods in biology. M., science, 1963, 272 pp.] And according to the Spearman rank correlation coefficient [Gubler EV, Genkin AA Application of nonparametric statistical criteria in biomedical research. L., Medicine, 1973, 142 pp.].

по сравнению с исходной, обусловленный увеличением доли клеток с интенсивностью флуоресценции более 100 усл.ед. (N_F>100), причем степень этого нарастания коррелировала с увеличением N_F>100. Через 5 часов хранения начиналось снижение $$\tilde{F}$$

из-за отсутствия питательных веществ в среде хранения, что также коррелировало со снижением N_F>100. Показано, что чем выше N_F>100 через 3-3,5 часа хранения, тем больше прирост концентрации ядросодержащих клеток в суспензии, который начинался через 5 часов хранения, что свидетельствовало о способности клеток к делению. При возрастании N_F>100 через 3-3,5 часа хранения ККМ менее чем в 1,5 раза по сравнению с исходной величиной, концентрация ядросодержащих клеток практически не изменялась, что свидетельствовало о неспособности клеток к делению в данных условиях.As a result of statistical processing of the obtained data, it was found that after 3-3.5 hours of storage, an increase was observed $$\tilde{F}$$

compared with the original, due to an increase in the proportion of cells with fluorescence intensity of more than 100 conventional units (N _{F> 100} ), and the degree of this increase correlated with an increase in N _{F> 100} . After 5 hours of storage, the decline began $$\tilde{F}$$

due to lack of nutrients in the storage medium, which also correlated with a decrease in N _{F> 100} . It was shown that the higher N _{F> 100} after 3-3.5 hours of storage, the greater the increase in the concentration of nucleated cells in suspension, which began after 5 hours of storage, indicating the ability of cells to divide. With an increase in N _{F> 100} after 3-3.5 hours of storage of CMC by less than 1.5 times compared to the initial value, the concentration of nucleated cells remained virtually unchanged, which indicated the inability of cells to divide under these conditions.

The method can be carried out, for example, as follows.

The suspension of KKM is carried out according to the published method [Fregatova L.M., Zubarovskaya L.S., Estrina M.A., Golovacheva A.A., Babenko E.V., Platonova G.G., Zueva E.E., Afanasyev B.V. Methods of obtaining stem cells in patients and donors for their subsequent transplantation. SPb, Publ. St. Petersburg State Medical University, Department of Hematology, Transfusiology, Transplantology FPO, 2004]. The resulting cells are placed in a standard stabilizer solution and stored at room temperature.

A cell suspension with a concentration of (~ 2-3) × 10 ⁷ with a volume of 0.9-1.0 ml is stored in 1.5 ml Eppendorf tubes at room temperature. Immediately after placing the cells in a test tube, the suspension is mixed and a sample of 30.0 μl is taken from it, placed in a 0.6 ml Eppendorf tube, a 2-Di-1-ASP solution is added at a final concentration of 1 · 10 ^-5 -1 · 10 ^-4 M and incubated for 45-90 minutes at 37 ° C. After incubation from the suspension of stained cells, a drop (3 μl) of the suspension is taken, placed on a defatted glass slide, covered with a coverslip and the cell preparation is examined using a Lumam-I2 fluorescence microscope (LOMO, Russia). The fluorescence of the probe is excited by a DRSh-250-2 mercury lamp with a wavelength of 405-436 nm. To register fluorescence, an FMEL-1 photometric nozzle and an interference filter with a transmission maximum of 585 nm are used. The excitation wavelength is 470 nm, the fluorescence intensity is determined at a wavelength of 560 nm. The fluorescence intensity of each individual cell is recorded. In each preparation, the fluorescence of an array of 80-100 cells is determined, N _{F> 100} is calculated in the original sample (storage time - 0 hours).

After 3-3.5 hours of storage of the CCM suspension from a test tube with a capacity of 1.5 ml, a second sample of 30.0 μl was taken after stirring, placed in a test tube with a capacity of 0.6 ml, a solution of 2-Di-1-ASP was added, incubated at 37 ° C and, after preparation of the preparation of stained cells, examine it on a luminescent microscope, calculate N _{F> 100} . With an increase in N _{F> 100} after 3-3.5 hours of storage, the CMC is more than 1.5 times compared with the initial value, the ability of the CMC to divide is judged.

The method is illustrated by the following clinical examples.

(средняя величина флуоресценции 80-100 индивидуальных клеток), рассчитывали N_F>100. Для контроля способности к делению определяли количество ядросодержащих клеток. Результаты исследования представлены в таблице 1.Example 1. Donor 1. KKM was stored in a standard stabilizer at room temperature. At certain time intervals, cell samples were taken, incubated with 2-Di-1-ASP at 37 ° C, determined $$\tilde{F}$$

(average fluorescence value of 80-100 individual cells), N _{F> 100 was} calculated. To control the ability to divide, the number of nucleated cells was determined. The results of the study are presented in table 1.

Table 1 Parameter Storage time, hour Ref. 3 5 24 48 72 R

усл.ед. $$\tilde{F}$$

conv.ed 81.05 ± 3.70 126.8 ± 4.4 * 118.3 ± 3.4 * 88.5 ± 2.7 ** 84.03 ± 2.50 ** 75.7 ± 3.0 ** N _{F> 100} % 26.6 67.2 45.8 25.0 36.3 23.1 0,025 # NC,% one hundred 115 170 209 190 200 * P = 0.05; ** P = 0.01 (t student test), comparison with $$\tilde{F}$$

ref. # - correlation between $$\tilde{F}$$

and N _{F> 100} (Spearman rank correlation coefficient).

в 1,56 раза. Через 5 часов хранения при снижении доли клеток с F>100 наблюдается увеличение концентрации ядросодержащих клеток в 1,7 раза по сравнению с исходной, что свидетельствует об их интенсивном делении. Через 24 часа величина N_F>100 практически возвращается к исходной, однако концентрация клеток продолжает нарастать (в 2 раза выше исходной). Через 72 часа N_F>100 ниже исходного уровня, но концентрация клеток по-прежнему в 2 раза выше исходной.The data obtained show that in the sample taken after 3 hours of storage, N _{F> 100} increases compared to the original 2.5 times, which correlates with the increase $$\tilde{F}$$

1.56 times. After 5 hours of storage, with a decrease in the proportion of cells with F> 100, an increase in the concentration of nucleated cells by 1.7 times in comparison with the initial one is observed, which indicates their intensive division. After 24 hours, the value of N _{F> 100} almost returns to the original, but the concentration of cells continues to increase (2 times higher than the original). After 72 hours, N _{F> 100 is} below the baseline, but the cell concentration is still 2 times higher than the baseline.

Thus, an increase in N _{F> 100} after 3 hours of storage of CMC by 2.5 times indicates their ability to divide.

, рассчитывали N_F>100, определяли количество ядросодержащих клеток. Результаты исследования представлены в таблице 2.Example 2. Donor 2. KKM was stored in a standard stabilizer at room temperature. At certain time intervals, cell samples were taken, incubated with 2-Di-1-ASP at 37 ° C, determined $$\tilde{F}$$

, N _{F> 100} was calculated, the number of nucleated cells was determined. The results of the study are presented in table 2.

table 2 Parameter Storage time, hour Ref. 3,5 5 24 48 72 R

усл.ед. $$\tilde{F}$$

conv.ed 82.03 ± 4.2 107.1 ± 3.9 * 97.5 ± 3.8 * 63.6 ± 4.0 ** 74.1 ± 2.9 ** 64.5 ± 3.3 ** N _{F> 100} % 31,0 53.3 38,0 21.6 16,2 10.0 0,025 # NC,% one hundred 103 150 160 150 150 * P = 0.05; ** P = 0.01 (t student test), comparison with $$\tilde{F}$$

ref. # - correlation between $$\tilde{F}$$

and N _{F> 100} (Spearman rank correlation coefficient).

возрастает в 1,3 раза). Через 5 часов при снижении N_F>100 наблюдается увеличение концентрации ядросодержащих клеток в 1,5 раза по сравнению с исходной, что свидетельствует об их делении. Через 24 часа N_F>100 ниже исходной, но концентрация клеток продолжает нарастать (в 1,6 раза выше исходной). Далее, до 72 часов концентрация клеток сохраняется практически на одном уровне.From the data of table 2 it can be seen that after 3.5 hours of storage, N _{F> 100} increases compared to the original 1.7 times ( $$\tilde{F}$$

increases by 1.3 times). After 5 hours, with a decrease in N _{F> 100} , a 1.5-fold increase in the concentration of nucleated cells is observed compared to the initial one, which indicates their division. After 24 hours, N _{F> 100 is} lower than the original, but the cell concentration continues to increase (1.6 times higher than the original). Further, up to 72 hours, the cell concentration remains almost at the same level.

From this example, it can be seen that an increase in N _{F> 100} after 3.5 hours of storage of CMC by 1.7 times indicates their ability to divide.

, рассчитывали N_F>100 определяли количество ядросодержащих клеток. Результаты исследования представлены в таблице 3.Example 3. Donor 3. KKM was stored in a standard stabilizer at room temperature. At certain time intervals, cell samples were taken, incubated with 2-Di-1-ASP at 37 ° C, determined $$\tilde{F}$$

, N _{F> 100 was} calculated; the number of nucleated cells was determined. The results of the study are presented in table 3.

Table 3 Parameter Storage time, hour Ref. 3 5 24 48 72 R

усл.ед. $$\tilde{F}$$

conv.ed 67.4 ± 2.5 79.0 ± 4.6 ** 58.8 ± 2.2 ** 59.2 ± 3.3 32.4 ± 2.3 * 35.6 ± 2.8 * N _{F> 100} ,% 22.3 26.5 14.3 7.0 1,5 0,0 0.05 # NC,% one hundred one hundred 101.7 102.3 one hundred one hundred * P = 0.05; ** P = 0.01 (t student test), comparison with $$\tilde{F}$$

ref. # - correlation between $$\tilde{F}$$

and N _{F> 100} (Spearman rank correlation coefficient).

- в 1,17 раза. Через 5 часов N_F>100 снижена в 1,6 раза по сравнению с исходной, нарастания концентрации ядросодержащих клеток нет, что свидетельствует об отсутствии деления клеток. Через 24 часа N_F>100 продолжает снижаться (в 3,5 раза по сравнению с исходной), общая концентрация клеток не изменяется. Через 72 часа в пробе отсутствуют клетки с интенсивностью флуоресценции более 100 усл.ед., деления клеток не происходит.From table 3 it can be seen that after 3 hours of storage in this preparation, N _{F> 100} increases by 1.2, and $$\tilde{F}$$

- 1.17 times. After 5 hours, N _{F> 100 was} reduced 1.6 times compared to the initial one; there was no increase in the concentration of nucleated cells, which indicates the absence of cell division. After 24 hours, N _{F> 100} continues to decrease (3.5 times compared with the original), the total concentration of cells does not change. After 72 hours, the sample contains no cells with a fluorescence intensity of more than 100 conventional units, cell division does not occur.

This example shows that the absence of an increase in N _{F> 100} after 3 hours of storage of CMC by 1.5 times indicates the inability of cells to divide.

Using the claimed method allows to evaluate the spontaneous ability of CMC to dividing, due to the effect of their own growth factors, and eliminates the reasons that affect cell division and differentiation. The method is methodologically simple and allows you to get results in 3-3.5 hours.

Claims (1)

A method for determining the ability of bone marrow cells to divide, characterized in that the cells are stored in a standard stabilizer solution at room temperature, the first initial sample is taken from the suspension, and after 3-3.5 hours of suspension storage, the second sample is taken, the samples are treated with a fluorescent potential-sensitive vital probe iodide 2- (p-dimethylaminostyryl) -1-methylpyridinium, measure the fluorescence intensity of the probe in the cells, calculate the proportion of cells with a fluorescence intensity of more than 100 conventional units in each sample, and with an increase in the proportion of cells with fluorescence intensity of more than 100 conventional units more than 1.5 times in the second sample compared with the first, they judge the ability of bone marrow cells to divide.