The transplantation of stem cells can be performed in an autologous, where the recipient donates his own stem cells for later use, or in an allogeneic fashion, where the donor and the recipient are two different persons (Berz, McCormack, Winer, Colvin, & Peter J Quesenberry, 2007; A. Gratwohl et al., 2010). In some clinical settings, stem cells can be utilized within a 72 hour timeframe without the need for extensive storage (Fleming & A Hubel, 2006; Allison Hubel, Carlquist, Clay, & Jeff McCullough, 2004; Pettengell, Woll, O'Connor, Dexter, & Testa, 1994). The autologous transplantation of cellular products and the therapeutic use of umbilical cord stem cells rely heavily on the preservation of stem cells after initial collection to be utilized at a later point in time (Berz, McCormack, Winer, Colvin, & Peter J Quesenberry, 2007). Hematopoietic stem cell transplantation has been in clinical use for decades to treat benign and malignant hematologic and non-hematologic conditions (C. J. Hunt, 2011). The principal sources for those therapeutic strategies are bone marrow, peripheral blood hematopoietic progenitor cells and stem cells derived from umbilical cord blood. Although clinicians have decades of experience with the use of hematopoietic stem cells(HSC), the interest in the clinical use of non-hematopoietic stem cells, such as embryonic(C. J. Hunt, 2011; Leeb et al., 2011), mesenchymal (Ding, Shyu, & Lin, 2011; Leclerc et al., 2011; Puglisi et al., 2011) and induced pluripotent stem cells (Leeb et al., 2011; Sohni & Verfaillie, 2011) is rapidly expanding. The therapeutic strategies utilizing such cellular products, depend heavily on the effective preservation of those cell products for clinical use at a later point in time. The need for ready availability of such products calls for storage procedures with favorable graft survival rates and a tolerable toxicity profile. While cryopreservation protocols for HSC are well developed, the field for non-hematopoietic stem cells still remains to be defined. The cryopreservation of all stem cells follows certain principal steps; First, Cytoreduction and prefreezing processing of the freshly collected graft. Second, the cryopreservative medium is prepared and added. Third, the graft is assessed for viability and integrity. Microbial contamination is ruled out. Fourth, freezing of the stem cells is performed. Fifth, thawing of the cryopreserved graft is conducted. Last, the post thawing processing is

/pdf/cryopreservation-of-hematopoietic-and-non-hematopoietic-stem-3svxa8uoog.pdf

Cryopreservation of Hematopoietic and Non-Hematopoietic Stem Cells – A Review for the Clinician

Background Banking of hematopoietic stem cells (HSCs) is a rapidly growing part of the transplant field. The essence of the banking process is to maintain the optimal quality parameters throughout the storage period, allowing successful transplantation. Methods Our laboratory research was carried out on 126 HSC samples that were collected by means of leukapheresis from patients with lymphoproliferative diseases. The samples were frozen in a controlled rate and stored up to 76 months in containers in vapor phase of liquid nitrogen. The evaluation was performed after thawing the probes. Viability of nuclear cells was assessed after incubation in Trypan blue, CD34+ phenotype cells were determined by means of cytometry with the use of 7-aminoactinomycin D (7-AAD), and an analysis of the proliferative potential of granulocyte-monocyte precursors was performed. For comparative statistical analysis, the material was divided into 3 groups according to storage time: A:  12 months (n = 31). Results In the examined groups, similar median values were observed of nuclear cell viability (A, 86%; B, 87%; and C, 83%) and CD34+ cells (95%, 94.5%, and 95.8%, respectively). A gradual, nonsignificant, reduction in the median of granulocyte-monocyte precursors was found: 68 × 104/kg of body weight (kg bw), 48.5 × 104/kg bw, and 47 × 104/kg bw, respectively. Statistical analysis with the use of the Kruskal-Wallis test showed a P value of >.05 for all variables. Conclusions There were no significant differences in the viability of nuclear cells, CD34+ cells, and proliferative potential granulocyte-monocyte precursors between groups. Storage for up to 76 months does not change the essential quality parameters, and HSCs could be qualified for distribution.

Banking of Hematopoietic Stem Cells: Influence of Storage Time on Their Quality Parameters.

Objectives Cryopreservation of hematopoietic stem cells intended for autologous transplantation is a crucial element of the banking process. Although cryopreservation techniques are well known, improvement is needed. This study was designed to optimize cryopreservation to improve the quantitative and qualitative parameters of hematopoietic stem cells in the material intended for transplantation. We used available opportunities to provide the best quantitative and qualitative parameters of hematopoietic stem cell transplants processed in a closed system. Material and methods Two hundred forty-eight products of hematopoietic stem cells collected by leukapheresis from patients with lymphoproliferative disorders create the basis of this report. The material was frozen in a controlled-rate freezer and stored in containers in the vapor phase of LN2 (−160°C). The composition of a cryoprotectant medium was modified. For freezing, 192 probes were used with a cryoprotective medium containing 20% dimethyl sulfoxide (DMSO) and enriched RPMI 1640. For 56 samples, we used 20% DMSO in autologous plasma harvested during leukapheresis. Products of hematopoietic stem cells and cryoprotectant medium were combined in a 1:1 ratio. The final number of nuclear cells did not exceed 2 × 108/mL. Analysis was performed after thawing the probes. Viability of nuclear cells has been assessed using the microscopic technique after incubation in Trypan blue and the CD34+ cells by flow cytometry using the 7-aminoactynomycin D. A statistical analysis has been conducted using the Statistica program (StatSoft, Cracow, Poland). Results and conclusions The results show that the application of autologous plasma is linked with higher viability of nuclear cells and CD34+ cells. Moreover, statistical analysis of the nuclear cells and CD34+ cells viability differs significantly between groups frozen using RPMI 1640 and autologous plasma (P

Analysis of the Effect of Cryoprotectant Medium Composition to Viability of Autologous Hematopoietic Cells Collected by Leukapheresis

The peripheral blood is a major source of hematopoietic stem cells. Almost for two decades the peripheral blood has been mobilized, in order to enhance the CD34+ concentration. The isolated stem cells from the mobilized peripheral blood are used as an alternative, or in addition to bone marrow derived stem cells. In this paper, a new perspective is being discussed; the use of non-mobilized peripheral blood as an alternative source for hematopoietic progenitor cells. The number of isolated hematopoietic stem cells is evaluated using flow cytometry. The viability can be evaluated using the trypan blue exclusion test, the flow cytometry or automated assays. The isolated hematopoietic stem cells could be used for ex vivo expansion either in static systems or in proper bioreactor systems, prior to cryopreservation and/or transplantation.

https://file.scirp.org/pdf/Health20100600001_75330063.pdf

Hematopoietic stem cells from peripheral blood the perspective of non-mobilized peripheral blood

/pdf/comparacao-da-viabilidade-das-celulas-mononucleares-totais-276lsnbje6.pdf

Comparação da viabilidade das células mononucleares totais da medula óssea de suínos em diferentes protocolos de congelamento

Freezing and thawing of hemopoietic stem cells (CD34+) are indispensable stages between their collection from peripheral blood or bone marrow and transfusion to the patient who has previously undergone myeloablative chemotherapy. At present there are two methods of the cells freezing: non-controlled--placing CD34+ cells directly in the final temperature and rate-controlled--gradual cooling them at programmed speed. Non-controlled freezing leads to the considerable loss of cells viability (up to 50%), but it doesn't require the expensive equipment used for rate-controlled freezing (viability loss up to 10%). In order to reduce the loss resulting from intra-cellular crystallization of water the haemopoietic cells are mixed with one of the cryopreservative substances: dimethylsulphoxide-DMSO, hydro-xyethylstarch-HES, polyvinylpyrrolidone-PVP or glycerol. The most important moment of freezing procedure is the phase transition of water. The adequate shape of the cooling curve leads to a considerable reduction of the loss of the cells viability. Further cooling is the most effective when it takes place at max. speed of 5 degrees C/min. The storage of the frozen cells is the best in very low temperatures (-170-180 degrees C-vapour phase of liquid nitrogen), but mechanical freezers (-80 degrees C) are used, too. The thawing procedure should be very fast (up to 90 degrees C/min.) and the defrosted cells must be immediately transfused to the patient because of very high toxicity of cryopreservative agents to non-frozen cells. The cells viability estimation is carried out with trypan blue or cytofluorometrically after incubation with propidine iodide.

[Methods for freezing, thawing and viability estimation of hemopoietic stem cells].

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[Methods for freezing, thawing and viability estimation of hemopoietic stem cells].