Evaluation of cryopreserved murine and human hematopoietic stem and progenitor cells designated for transplantation

Abstract

The influence of five different cryopreservation protocols on the quality and/or quantity of frozen cells was investigated on mouse bone marrow cells and human peripheral blood mononuclear cells (MNC). The efficiency of the protocols was evaluated on the basis of the recovery of very primitive pluripotent hematopoietic stem cells (MRA), pluripotent progenitors (CFU-Sd12), committed granulocyte-monocyte progenitors (CFU-GM) of mouse cells after thawing. The recovery of MRA, CFU-Sd12 and CFU-GM varied depending on the type of freezing procedure and cryoprotector (DMSO) concentrations used. It was shown that the controlled-rate protocol was more efficient, enabling better recovery of all categories of progenitor cells in frozen samples. The most efficient was the controlled-rate protocol of the cryopreservation designed to compensate for the release of fusion heat, which enabled the best recovery of CFU-GM (73.0±8.8%) and CFU-Sd12 (90.0±15.9%) when combined with 5% DMSO concentration (protocol 4). On the contrary, a better recovery (79.8±13.5%) of very primitive stem cells (MRA) was achieved only when the higher concentration (10%) DM SO was used in combination with a five-step protocol of cryopreservation (protocol 1). These results pointed out the adequately used controlled-rate freezing to be essential for a highly efficient cryopreservation of some of the categories of hematopoietic stem and progenitor cells. A t the same time, it was obvious that a higher DMSO concentration was necessary for the cryopreservation of MRA, but not for more mature progenitor cells (CFU-S, CFU-GM). These results imply the existence of a mechanism that decreases the intracellular concentration of DMSO in MRA cells, which is not the case in less primitive progenitors. For human MNC, the recovery and viability of the cells, as well as the engraftment potential of cryopreserved cells after thawing were investigated. Cryopreservation protocol 1 resulted in better MNC recovery (82.7±10.4%) than protocol 3 (49.9 ±15.1%). The mean recovery of MNCs (collected from patients for autologous transplantation) was 78.5±7.3% (protocol 1) and 53.1 ±26.2% (protocol 3). The obtained favorable recovery of thawed cells and rapid reconstitution of hematopoiesis (on the day 11th following the transplantation) in patients confirmed that the controlled-rate freezing in combination with optimal DMSO concentration was able to obtain sufficient progenitor cryoprotection.

Efikasnost pet protokola kriokonzervacije ispitivana je na osnovu oporavka ćelija kostne srži miševa i humanih mononuklearnih ćelija periferne krvi (MNC) posle odmrzavanja. Kod miševa, određivan je oporavak veoma primitivnih pluripotentnih hematopoeznih progenitora (MRA), pluripotentnih i opredeljenih progenitora (CFU-GM i CFU-Sd12). Oporavak MRA, CFU-Sd12 i CFU-GM varirao je u zavisnosti od vrste primenjene procedure zamrzavanja i koncentracije dimetil sulfoksida (DMSO). Pri izvođenju procedure programiranog zamrzavanja konstatovan je bolji oporavak svih kategorija progenitora. Programirano zamrzavanje sa kompenzacijom oslobođene toplote fuzije i upotrebom 5% DMSO (protokol 4) omogućilo je najbolji oporavak CFU-GM (73,0±8,8%) i CFU-Sd12 (90,0±15,9%). Suprotno tome, oporavak veoma primitivnih MRA bio je bolji (79,8±13,5%) ukoliko je primenjena veća koncentracija (10%) DMSO u kombinaciji sa petostepenom procedurom zamrzavanja (protokol 1). Ovi rezultati ukazuju na neophodnost primene programiranog zamrzavanja pri kriokonzervaciji pojedinih kategorija hematopoeznih progenitora. Utvrđeno je da efikasna kriokonzervacija MRA, ali ne i manje primitivnih progenitora (CFU-S, CFU-GM), zahteva upotrebu veće koncentracije DMSO. Sve to ukazuje na moguće postojanje nekog mehanizma koji izaziva redukciju intracelularne koncentracije DMSO u ćelijama MRA, ali ne i u manje primitivnim progenitorima. Kod humanih MNC, određivan je oporavak ćelija nakon odmrzavanja i stepen rekonstitucija hematopoeze nakon autologne transplantacije. Kriokonzervacija po protokolu 1 je rezultovala boljim oporavkom humanih MNC (82,7±10,4%) od protokola 3 (49,9±15,1%). Prosečni oporavak MNC (prikupljenih od bolesnika za autolognu transplantaciju) iznosio je 78,5±7,3% (protokol 1), odnosno 53,1±26,2% (protokol 3). Postignuti visoki stepen oporavka odmrznutih ćelija i brza rekonstitucija hematopoeze (11. dan posle transplantacije) kod ispitivanih bolesnika su potvrdili da programirano zamrzavanje u kombinaciji sa optimalnom koncentracijom DMSO omogućava efikasnu zaštitu progenitora od kriooštećenja.