Collection, In Vitro Expansion, and Freezing of Human Stem Cells of Fetal Origin.
Authors: Machaj Eugeniusz K. , Jastrzewska Marzena , Gajkowska Agnieszka , Oldak Tomasz , Debski Romuald , Pojda Zygmunt 
Published: April 2020

Abstract
Fetal stem cells have several therapeutical advantages when compared to their adult counterparts. Cell populations residing in “waste” tissues (cord blood, umbilical cord and placenta) may be collected without any medical or ethical contraindications concerning mother or newborn baby. Cord blood hematopoietic stem cells are routinely used for clinical transplantations, and the methods for their collection, isolation and freezing are now well standardized. We have tested the efficiency of cell separator (Sepax, Biosafe) in volume reduction of cord blood units, and in washing-out DMSO after cell thawing. Results of the volume-reduction procedure revealed that the average MNC recovery rate was 91±5%, cell viability was ≥90%, and CD34+ cell recovery rate was 92±4%. Volume reduction process eliminated 88±4.5% of red cells. In vitro clonogenic potential of cells isolated with Sepax did not differ from this of cells isolated according to the standard HES sedimentation protocol. Protocol of Sepax-based washing of thawed cells resulted of 9±4% cell loss and 10±6% decrease in cell viability, when compared to thawed non-processed cells. We conclude, that Sepax separator may be used both for cord blood volume reduction, and DMSO elimination from thawed samples.
The other cord blood stem cell population, identified by us (J Cell Sci 2002, 115:2131–2138), was plastic-adherent, nonhematopoietic stem cell differentiating at least into neuropoietic progeny. Attempted optimisation of cell isolation/culture methods did not allow for successful isolation of these cells from more than approximately 20% cord blood samples - we postulate, that this cell population, when detected in mature newborn blood, is rather a remnant of cells circulating in earlier stages of fetus development.
The population of cells residing in Wharton jelly of umbilical cord (CD34−, CD45−, CD29+, CD44+, CD51+, CD105+, SH-2+, SH-3+, plastic-adherent) is capable, according to published data, to differentiate into several tissues, resembling the adult mesenchymal stem cells. We were able to isolate these cells from 85% of cord collections, and to expand them for 5–10 passages in in vitro cultures in IMDM + 10% FCS media without growth factors added. We have also developed the protocol, allowing for storage of frozen umbilical cord tissue without preliminary cell isolation and expansion, allowing for labor-economical and cost-economical banking of large numbers of umbilical cord cell samples.
Conclusions:
Sepax application for cord blood volume reduction, or DMSO elimination from thawed cell samples, is an improvement in cord blood bank routine protocols. The low frequency of non-hematopoietic plastic-adherent stem cells in cord blood samples, collected during mature deliveries, is rather a biological phenomenon than the inadequacy of laboratory techniques, and may reduce the utility of these cells for clinical purposes. Stem cells from Wharton’s jelly of umbilical cords, if confirmed their “stemness”, can be an easy to obtain cell source for tissue engineering. Our technique of freezing-storage of the umbilical cord tissue may be useful for low-cost, time-saving banking of large numbers of fetal stem cells.