Supplementation of Conventional Freezing Medium with a Combination of Catalase and Trehalose Results in Better Protection of Surface Molecules and Functionality of Hematopoietic Cells

Abstract

Our previous studies had shown that a combination of the bio-antioxidant catalase and the membrane stabilizer trehalose in the conventional freezing mixture affords better cryoprotection to hematopoietic cells as judged by clonogenic assays. In the present investigation, we extended these studies using several parameters like responsiveness to growth factors, expression of growth factor receptors, adhesion assays, adhesion molecule expression, and long-term culture-forming ability. Cells were frozen with (test cells) or without additives (control cells) in the conventional medium containing 10% dimethylsulfoxide (DMSO). Experiments were done on mononuclear cells (MNC) from cord blood/fetal liver hematopoietic cells (CB/FL) and CD34(+) cells isolated from frozen MNC. Our results showed that the responsiveness of test cells to the two early-acting cytokines, viz. interleukin-3 (IL-3) and stem cell factor (SCF) in CFU assays was better than control cells as seen by higher colony formation at limiting concentrations of these cytokines. We, therefore, analyzed the expression of these two growth factor receptors by flow cytometry. We found that in cryopreserved test MNC, as well as CD34(+) cells isolated from them, the expression of both cytokine receptors was two- to three-fold higher than control MNC and CD34(+) cells isolated from them. Adhesion assays carried out with CB/FL-derived CD34(+) cells and KG1a cells showed significantly higher adherence of test cells to M210B4 than respective control cells. Cryopreserved test MNC as well as CD34(+) cells isolated from them showed increased expression of adhesion molecules like CD43, CD44, CD49d, and CD49e. On isolated CD34(+) cells and KG1a cells, there was a two- to three-fold increase in a double-positive population expressing CD34/L-selectin in test cells as compared to control cells. Long-term cultures (LTC) were set up with frozen MNC as well as with CD34(+) cells. Clonogenic cells from LTC were enumerated at the end of the fifth week. There was a significantly increased formation of CFU from test cells than from control cells, indicating better preservation of early progenitors in test cells. Our results suggest that use of a combination of catalase and trehalose as a supplement in the conventional freezing medium results in better protection of growth factor receptors, adhesion molecules, and functionality of hematopoietic cells, yielding a better graft quality.

Cryopreservation of Hematopoietic and Non-Hematopoietic Stem Cells – A Review for the Clinician

“…Cell count recovery, viability and clonogenicity were similar in several studies (Rodrigues et al, 2008). Similarly encouraging results have been noticed in the presence of catalase type natural biooxidants in the cryopreservation medium (Motta, Gomes, L F Bouzas, Paraguassú-Braga, & Porto, 2010;Sasnoor, Kale, & Limaye, 2003), although the exact role of the natural biooxidant is still not known. Another group of substances which are excellent candidates as cryo-preservative additives are hydrophilic macromolecules.…”

Proteins in Saccharides Matrices and the Trehalose Peculiarity: Biochemical and Biophysical Properties

“…Trehalose combined with catalase was used as a supplement in the conventional freezing medium of hematopoietic cells and gave a better protection of functionality, hence a better graft quality, in particular for growth factor receptors and adhesion molecules [73].…”

Preservation of stem cells

“…24 Other investigators have studied the use of additives (in addition to DMSO) designed to improve post thaw recovery of HSCs. Low levels of trehalose (25 mg/ml) and catalase (100 μg/ml) were shown to reduce post thaw apoptosis in a murine HSC model 25 and improve recovery of colony forming units for umbilical cord blood and HSCs isolated from fetal liver. 26 Cryopreservation solutions supplemented with membrane stabilizer taurine, ascorbic acid and α-tocopheryl acetate also demonstrated improved post thaw recovery.…”