1 research outputs found
Transient warming events and cryogenic
Background: The term “Transient Warming Events” (TWE) has been used to describe the
effects of brief periods of exposure of cryopreserved cells to ambient temperature in the course of
retrieving and returning outgoing samples from the same cane. This study was designed to
evaluate the effect of multiple TWEs on cryogenically preserved umbilical cord blood units
(UCB) at the New Jersey Community Blood Centre using established assays of cell recovery,
viability, and clonogenicity.
Materials and methods: The UCBs were collected and processed according to the laboratory
SOPs from consenting donors. Each UCB was split into two 25 mL samples: an experimental
and a control sample. Both samples were frozen using a Controlled Rate Freezer and then
stored below –180掳C in the vapour phase of a liquid nitrogen (LN2) freezer. The experimental
samples were exposed 10 times to room temperature until the sample reached a target temperature.
We tested target temperatures of –110掳C and –80掳C. Finally, both experiment and control
samples were simultaneously thawed in a 37掳C dry bath. Each sample was tested for Total
Nucleated Cell (TNC) count, CD34+ cell count, cell viability, and ability to generate Colony
Forming Units (CFU) in a standardized assay.
Results: When UCB units were each exposed to 10 TWE to a target temperature of –110掳C,
analysis of cell counts showed a 1.5% loss of TNC, 3.5% loss of CD34+ cells, and 2% drop in
CFU, with no loss of viability. None of the values between experimental and control was statistically
significant. When UCB units were exposed to 10 TWE to a target temperature of –80掳C, the
differences between experimental and control samples in the TNC, CD34+ cell count and viability
were also not statistically significant. The numbers of CFUs in experimental samples was decreased
by 8% compared to control, and reached statistical significance. In the New Jersey Community
Blood Centre, removal of a UCB from cryogenic storage takes less than a minute to execute,
typical of other UCB storage centres with well planned and executed sample retrieval protocols. It
would take three times as long for a 25 ml UCB to reach a target temperature of –110掳C, and five
times as long to reach a target temperature of –80掳C. We found no significant degradation of cell
function after repeated TWE to target temperatures as high as –110掳C.Conclusions: The results of this study demonstrate that the TWE that UCB units experience
in day-to-day sample retrieval activities are not detrimental to the samples that are returned
repeatedly to cryogenic storage at the New Jersey Community Blood Centre. This should
encourage other centres to conduct their own TWE studies to evaluate the effects of TWEs with
each type of cryopreserved tissue and associated methods of storage and retrieval, before considering
investing in highly expensive cryostorage units that are designed to minimize TWEs.Wst臋p: Okre艣lenie Transient Warming Events (TWEs) oznacza kr贸tkie okresy ekspozycji
zamro偶onych kom贸rek na temperatur臋 otoczenia, w trakcie rutynowych czynno艣ci laboratoryjnych.
Celem pracy by艂a ocena wp艂ywu wielokrotnych TWEs na wybrane parametry zamro偶onych
jednostek krwi p臋powinowej (UCB) przechowywanych w New Jersey Community Blood
Center (NJCBC). Wykorzystano znane metody oceny i odzysku kom贸rek, ich 偶ywotno艣ci
i zdolno艣ci klonogennych.
Materia艂 i metody: Jednostki UCB pobierano za zgod膮 dawc贸w i poddawano preparatyce
zgodnie z obowi膮zuj膮cymi procedurami (SOP, standard operating procedures). Ka偶d膮 z nich
dzielono na dwie 25-mililitrowe pr贸bki, mro偶ono w urz膮dzeniu do kontrolowanego zamra偶ania
i przechowywano < –180掳C. Pr贸bki badane poddawano 10-krotnej ekspozycji na temperatur臋
pokojow膮, a偶 do uzyskania temperatur docelowych (–110掳C , –80掳C). Pr贸bki kontrolne
przechowywano w sta艂ej temperaturze poni偶ej –180掳C. Nast臋pnie pr贸bki badane i kontrolne
rozmra偶ano r贸wnocze艣nie w urz膮dzeniu do suchego rozmra偶ania w temperaturze 37掳C.
W ka偶dej pr贸bce oceniano ca艂kowit膮 liczb臋 kom贸rek j膮drzastych (TNC, total nucleated cell
count), liczb臋 kom贸rek CD34+, 偶ywotno艣膰 kom贸rek oraz liczb臋 jednostek tworz膮cych kolonie
(CFU, colony forming units).
Wyniki: W pr贸bkach poddanych 10-krotnemu dzia艂aniu temperatury pokojowej a偶 do osi膮gni臋cia
docelowej temperatury –110掳C stwierdzono 1,5-procentowe zmniejszenie TNC,
3,5-procentowe zmniejszenie liczby kom贸rek CD34+ oraz 2-procentowe zmniejszenie ca艂kowitej
liczby CFU. Nie zaobserwowano strat w 偶ywotno艣ci kom贸rek. R贸偶nice mi臋dzy pr贸bkami
badanymi a kontrolnymi w zakresie badanych parametr贸w nie by艂y statystycznie istotne.
R贸偶nice pomi臋dzy pr贸bkami badanymi poddanymi 10-krotnemu dzia艂aniu temperatury pokojowej,
a偶 do osi膮gni臋cia temperatury –80掳C a pr贸bkami kontrolnymi r贸wnie偶 nie by艂y statystycznie
istotne pod wzgl臋dem liczby TNC, liczby kom贸rek CD34+ i 偶ywotno艣ci; ca艂kowita
liczba CFU w pr贸bkach badanych by艂a ni偶sza o 8%, co by艂o statystycznie istotne. W NJCBC
jednorazowa ekspozycja jednostek UCB na temperatur臋 pokojow膮 nie przekracza 1 min,
a uzyskanie temperatury –110掳C trwa oko艂o 3,5 minuty.
Wnioski: Wyniki naszych bada艅 wykazuj膮, 偶e wielokrotna, kontrolowana ekspozycja zamro偶onych
jednostek UCB w trakcie rutynowych czynno艣ci laboratoryjnych, nie wywiera szkodliwego
wp艂ywu na jako艣膰 biologiczn膮 preparat贸w. Uzyskane przez nas wyniki powinny zach臋ci膰
inne o艣rodki do prowadzenia w艂asnych bada艅 nad wp艂ywem TWE na jako艣膰 poszczeg贸lnych
rodzaj贸w kom贸rek przechowywanych w stanie zamro偶enia oraz stosowanych metod
zamra偶ania i rozmra偶ania. Dopiero w贸wczas mo偶na podejmowa膰 decyzje o zakupie wysoce
specjalistycznego sprz臋tu ch艂odniczego z zamiarem obni偶enia liczby TW