Evaluation of energy potential of fresh and stored bone marrow cells using a fluorescent potential-sensitive probe

Abstract

SummaryBone marrow is a primary source of hematopoietic stem cells in clinical transplantation. Quality of bone marrow grafts is a key factor of their successful in vivo expansion. The aim of our work was to test a semi-quantitative technique for assessment of bone marrow cell viability under strict storage conditions, by means of a fluorescent membrane potential-sensitive 2-Di-1-ASP probe.We have studied 20 samples of normal bone marrow cells (BMC). The cells were placed in a standard storage solution with sodium citrate, citric acid; phosphate salts, dextrose and adenine. Cell counts and viability tests were performed up to 72 hours of incubation. The samples were labeled with 2-Di-1-ASP probe at specified terms. Fluorescence intensity was measured for single nucleated cells, followed by calculating mean fluorescence values and myelokaryocyte numbers. Mitotic indexes were determined both in Giemsa-stained and fluorescent probe-stained cells. Cluster analysis and non-parametric tests were used for statistical evaluation. ResultsInitial cell survival of 80-92% was shown at 3…5 hours of storage, then decreased to 70-75% by the end of incubation. Meanwhile, cell incubation for 3 hours was accompanied by increased fluorescence, in terms of F̃ values, mainly, due to higher proportion of "bright" cell population (>100 arb.units, N F>100 ,%). D (ratio of N F>100 at 3h storage to N F>100 initial) proved to be the most informative parameter, thus enabling us to predict sample-specific differences for the F̃ values at later terms. All BMC samples exhibited increased F, on the account of brighter cell population (N F>100 ), over 3 hours of incubation. This increase correlated with increase in myelokaryocyte counts. An additional cluster analysis allowed us to classify the BMC samples into 3 sub-groups, by their significant inter-group differences for D values and cell number changes. In particular, a number of mitotic cells were detected in BMC populations at 5 to 24 hours of incubation, showing bright stainability with 2-Di-1-ASP probe. We revealed 0.60±0.10% of metaphase cells at initial time point. After 5-h storage, the frequency of mitotic cells increased to 1.4±0.1%; and, after 6-h colchicine treatment, the mitotic index increased to: 1.8±0.1%, thus showing good preservation of dividing cell fraction.In summary, our results have shown sustained, and even increased energy activity using a potential-sensitive probe, and good survival of mitotic cell fraction under strict incubation conditions. Appropriate mechanistic studies of the bone marrow cell preservation and energy balance under the given storage conditions should be performed in future.

Application of fluorescent potential-sensitive probes for the study of living blood and marrow cells

“…We have found optimal working conditions with the probe, i.e., incubation terms and temperature, probe concentration. It has been experimentally confirmed that the tests with 2-Di-1-ASP are register changes in the energy potential of BMC when exposed to various factors: cryopreservation with dimethyl sulfoxide, thawing procedure, suspension in saline solution, Hanks medium, standard stabilizer medium, storage duration [59].…”

“…A total of 9130 cells were examined in 500 preparations. It was found that, in all BMC samples, after 3-3.5 hours of storage at room temperature, an increased intensity of probe-specific fluorescence (F) was recorded [61].…”

“…Seventy to hundred cells per sample were tested manually, and the mean fluorescence intensity of single cells (F, arbitrary units) was calculated for each specimen. Photographic pictures of luminescent objects were performed with TCA-5.0 camera, using "Micro-Analysis View" software ("LOMO LLC Microsystems", Russia) [61].…”

Noninvasive method of monitoring the functional state of living cells

“…Найдены оптимальные условия работы с зондом: температура, время инкубации, концентрация зонда. Экспериментально подтверждено, что тесты с 2-Di-1-ASP регистрируют изменения энергетического потенциала ККМ при воздействии на них различных факторов: криоконсервации с диметилсульфоксидом, размораживания, суспендирования в физиологическом растворе, среде Хенкса, среде стандартного стабилизатора, длительности хранения [59].…”

“…Всего было исследовано 9130 клеток в 500 препаратах. В результате проведенных исследований было установлено, что во всех образцах ККМ через 3-3,5 часа хранения при комнатной температуре регистрировался рост интенсивности свечения зонда ( ∼ F) [61]. В публикации немецких авторов [62] обсуждался вопрос жизнеспособности трансплантатов ККМ и стволовых клеток, выделенных из периферической крови (ПСК) при различных температурных режимах.…”