Učinak kriopohrane na preživljenje krvotvornih matičnih stanica i subpopulacija leukocita prikupljenih iz periferne krvi postupkom leukafereze

doctoral thesis

• Title (english): The effect of cryopreservation on recovery of hematopoetic stem cells and leukocytes subpopulation collected from peripheral blood by leukapheresis
• Author: Vladimira Rimac
• Mentor: Ines Bojanić (mentor)
• Mentor: Sanja Dabelić (komentor)
• Committee member: Marija Grdić Rajković (predsjednik povjerenstva)
• Committee member: Irena Jukić (član povjerenstva)
• Committee member: Radovan Vrhovac (član povjerenstva)
• Granter: University of Zagreb; Faculty of Pharmacy and Biochemistry; (Department of biochemistry and molecular biology); Zagreb
• Defense date and country: 2023-12-13, Croatia
• Scientific / art field, discipline and subdiscipline: BIOMEDICINE AND HEALTHCARE; Pharmacy; Medical Biochemistry
• Universal decimal classification (UDC): 615 - Pharmacology. Therapeutics. Toxicology

Abstract

Zbog očekivanog daljnjeg razvitka i primjene pripravaka za staničnu terapiju, potrebno je poznavati čimbenike koji utječu na vijabilnost i preživljenje stanica nakon kriopohrane. Cilj ovog prospektivnog istraživanja bio je u uzorcima autolognih i alogenih pripravaka stanica prikupljenih iz periferne krvi leukaferezom odrediti broj živih krvotvornih matičnih stanica (KMS) i subpopulacija leukocita 7-amino aktinomicin D (7-AAD)/aneksin V metodom i standardnom 7-AAD metodom te ispitati utjecaj kriopohrane i različitih čimbenika na preživljenje odmrznutih stanica, a i vremensko razdoblje od odmrzavanja koje je prihvatljivo za određivanje vijabilnosti stanica u svrhu procjene kvalitete pripravaka stanica. U 30 autolognih i 30 alogenih uzoraka pripravaka stanica „single-platform“ metodom određen je broj živih KMS-a, limfocita T i B, NK stanica i monocita. Stabilnost odmrznutih uzoraka ispitana je u tri točke: odmah po odmrzavanju te sat vremena i tri sata nakon odmrzavanja. Vijabilnosti svih populacija stanica određene 7-AAD/aneksin V metodom bile su statistički značajno niže u odnosu na 7-AAD metodu u svježim i odmrznutim uzorcima autolognih i alogenih pripravaka, no dobivene razlike nemaju utjecaj na laboratorijsku procjenu kvalitete pripravaka KMS. U autolognim pripravcima kriopohrana najmanje utječe na preživljenje CD3+/CD8+ stanica, a u alogenima na limfocite B (CD19+ stanice). U autolognim i alogenim pripravcima kriopohrana najviše utječe na postotak preživljenja CD34+ stanica, a kod subpopulacija leukocita na preživljenje monocita (CD14+ stanica) i u autolognim i alogenim pripravcima. U autolognim pripravcima na preživljenje CD3+ i CD3+/CD4+ stanica negativno je utjecala viša koncentracija trombocita, a na preživljenje CD3+/CD8+ stanica duljina pohrane svježeg pripravka prije kriopohrane. U alogenim pripravcima postotak granulocita negativno je utjecao na preživljenje limfocita T.
Ispitivanjem utjecaja kriozaštitne otopine na vijabilnost stanica nakon odmrzavanja dobiveno je da se apsolutni broj živih KMS-a, limfocita B i monocita statistički značajno smanjuje već sat vremena od odmrzavanja te je pripravke s tim vrstama stanice potrebno transplantirati odmah po odmrzavanju.
U ovom istraživanju zapaženo je različito preživljenje KMS-a i subpopulacija leukocita nakon kriopohrane. Stoga bi svaki transplantacijski centar trebao u svojim uvjetima ispitati kako kriozaštitna otopina i ostali čimbenici utječu na vijabilnost i preživljenje stanica od interesa.

Abstract (english)

Due to the expansion of cell therapy using not only haematopoietic stem cells (HSC) but also other leukocyte subpopulations, the loss of these cells in cryopreserved apheresis products needs to be evaluated. The primary aim of this prospective study was to evaluate the post-thaw recovery of HSC and leukocyte subpopulations using the flow cytometry 7-AAD and 7-AAD/annexin V methods. The secondary aim was to assess how the post-thaw recovery is affected by various parameters, and the third aim was to examine how the time of exposure to cryoprotective solution containing DMSO after thawing affects the sample stability and viability of HCS and leukocyte subpopulations in autologous and allogeneic PBSC samples. In samples of 30 autologous and 30 allogeneic PBSC products, the number of viable HCS, T and B lymphocytes, NK cells and monocytes was determined using the "single-platform" method. The stability of the samples post-thaw was tested at three points: immediately after thawing and one hour and three hours post-thaw. The lowest post-thaw recovery was observed in autologous and allogeneic for CD34+ cells. In leukocyte subpopulation, the lowest post-thaw recovery was observed for CD14+ cells, both autologous and allogeneic. The highest post-thaw recovery was observed for CD3+/CD8+ cells in autologous, and for CD19+ cells in allogeneic samples. The statistically significant difference was observed between autologous and allogeneic PBSC products for CD3+ cell recovery and CD3+/CD8+ cell recovery. The evaluation of factors that could adversely affect the post-thaw recovery in autologous samples showed weak negative correlations between platelet concentration and CD3+ recovery, as well as between storage time and CD3+CD8+ recovery. In allogeneic samples, a strong negative correlation was observed only between the percentage of granulocytes and CD3+, CD3+/CD8+ and CD3+/CD4+ cell recoveries. Analysis of the absolute count of viable cells in thawed samples showed a significant difference between all measurement points for CD34+, CD14+ and CD19+ cells. No significant differences were observed for post-thaw stability of allogeneic samples analysed between products stored before cryopreservation ≥ 24 hours (N=20), and those stored < 24 hours (N=10), except for viable CD3+/CD4+ cells after three hours post-thaw. Since various post-thaw recoveries of leukocyte subpopulations were observed, the cell therapy manufacturing centers should evaluate how their cryopreservation method and other factors affect the recovery of cell population of interest in their settings.

• Keywords: stanična terapija; krvotvorne matične stanice; subpopulacije leukocita; kriopohrana; preživljenje stanica
• Keywords (english): cell therapy; peripheral blood stem cells; leukocyte subpopulations; cryopreservation; post-thaw recovery
• Language: croatian
• URN:NBN: urn:nbn:hr:163:717566
• Promotion: 2023
• Study programme: Title: Pharmacy and biochemistry; Study programme type: university; Study level: postgraduate; Academic / professional title: doktor znanosti (doktor znanosti)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Open access
• Created on: 2024-01-26 11:07:59