Cryogenic and low temperature preservation of human placental villous explants - a new way to explore drugs in pregnancy disorders.
BACKGROUND
A major handicap in cell culture studies using human tissues is the insufficient availability of fresh material on site. A method was developed for cryogenic storage and low temperature preservation of human placental villous explants, facilitating multi-site distribution for functional studies.


METHODS
Explants from term placentas were incubated with cryoprotectant agents (dimethyl-sulfoxide (DMSO), ethylene glycol, propanediol or Aedesta), frozen in liquid nitrogen, thawed and then cultured in-vitro. Viability was assessed by comparing frozen and thawed explants with non-frozen controls for morphological changes, lactate dehydrogenase (LDH) release, placenta protein 13 (PP13) secretion, and PCNA Western blotting. Functional studies determined the effect of oxygen and magnesium on explant viability.


RESULTS
Cryoprotection by 3 M DMSO best maintained explants' viability, morphological integrity and PP13 release after freezing and thawing from liquid nitrogen. The effect of oxygen and magnesium was used to test the functional viability of cultured explants, after freezing in liquid nitrogen and transfer to dry ice for 1-5 days on site or for shipment to a remote lab. The tested parameters were similar between controls and cryogenically treated explants in the remote lab and the lab of origin, demonstrating the possibility of cryostoring explants for functional studies.


CONCLUSIONS
Cryogenically stored placental villous explants shipped frozen can serve as a useful tool for comparative functional studies of placental villous tissues. The results of this pilot study also open the way for multi-site studies associated with drug tailoring for pregnancy disorders.
Institute of Cell Biology, Histology and Embryology, Medical University of Graz, Harrachgasse 21/7, 8010 Graz, Austria. berthold.huppertz@medunigraz.at
B
B
Huppertz
V
V
Kivity
M
M
Sammar
Y
Y
Grimpel
N
N
Leepaz
K
K
Orendi
I
I
Pekarski
H
H
Meiri
R
R
Gonen
E
E
Lubzens
D000328:Adult; D000465:Algorithms; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D002824:Chorionic Villi / Q000187:drug effects / Q000378:metabolism; D003080:Cold Temperature; D015925:Cryopreservation / Q000379:methods*; D003451:Cryoprotective Agents / Q000494:pharmacology; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D005260:Female; D037161:Galectins / Q000378:metabolism; D006801:Humans; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D011247:Pregnancy; D011248:Pregnancy Complications / Q000188:drug therapy* / Q000473:pathology* / Q000517:prevention & control; D011257:Pregnancy Proteins / Q000378:metabolism; D014021:Tissue Preservation / Q000379:methods*; D055815:Young Adult
D003451:Cryoprotective Agents; D037161:Galectins; C038195:LGALS13 protein, human; D011257:Pregnancy Proteins; D007770:L-Lactate Dehydrogenase