FREEZING RESPONSE AND POST FREEZE DIFFERENTIATION POTENTIAL OF HUMAN ADIPOSE DERIVED ADULT STEM CELL (huASCs)

This study presents volumetric shrinkage (water transport) during freezing of human adipose derived adult stem stem cells (huASCs) using a calorimetric technique in the presence and absence of cryoprotective agents (CPAs). By fitting a model of water transport to the experimentally obtained volumetric shrinkage data we determined the membrane permeability parameters (reference membrane permeability to water, Lpg or Lpg[cpa] and the activation energy, ELp or ELp[cpa]) [1-3]. The experimentally determined membrane permeability parameters were then used to calculate the optimal rates of freezing huASCs cells in the presence and absence of CPAs [1-3]. Additionally, we have investigated the ability of frozen-thawed huASCs to form a colony of osteoblasts using a colony-formation-unit (CFU) assay [4]. INTRODUCTION Human adipose tissue provides a uniquely abundant and accessible source of adult stem cells. In response to chemical, hormonal or structural stimuli, these human adipose-derived adult stem cells (huASCs) can differentiate along multiple lineage pathways, including adipocytes, chondrocytes, myocytes, neurons and osteoblasts [4]. Successful cryopreservation of scientifically and commercially important huASCs would revolutionize the tissue engineering and regenerative medicine industry. All cell systems do share common cryobiological responses, which may be exploited to better understand and alleviate the specific problems of freezing in huASCs cells. The highest rates of cellular survival are typically found for cooling rates which are fast enough to minimize dehydration solute effects injury while still slow enough to preclude large amounts of intracellular ice. Thus, to optimize and generate a firm biophysical understanding of the freezing process in any biological system, both water transport (dehydration) and intracellular ice formation (IIF) need to be experimentally determined. We are unaware of any studies that report water transport or IIF parameters in any stem cells. This study aims to rectify this lack of cryobiological knowledge in stromal vascular fraction (SVF) and several passages (P0, P2, and P4) huASCs stem cells using a DSC technique. A more detailed explanation of the DSC technique is given elsewhere [5]. WATER TRANSPORT MODEL Mazur [6] developed a mathematical model for the volumetric change in cells due to the water transport during freezing process in the presence of extracellular ice and CPAs. In this water transport model,   B CPAs, of Conc. and Type , , , , WV SA E L f dT dV Lp pg  ; where dV/dT is the rate of volumetric shrinkage of a cell, Lpg or Lpg[CPA] is the reference membrane permeability, ELp or ELp[CPA] is the activation energy, SA is the surface area available for water transport, WV is the initial volume of intracellular water and B is the imposed cooling rate ( ̊C/min). A detailed description of the water transport model is presented elsewhere [5, 6]. And finally a nonlinear least squares curve fitting technique was implemented in a computer program to calculate the best fit membrane permeability parameters (Lpg and ELp or Lpg[CPA] and ELp[CPA]), as previously described [1,2,5] RESULTS Fig 1 shows the water transport data (filled circles) and simulation ( ________ ) for P2 cells using the ‘best fit’ parameters in the water transport model at cooling rate 20 ̊C/min the absence of CPAs (Fig. 1A), in the presence of 10% glycerol (Fig. 1B) and in the presence of 10% DMSO (Fig. 1C). The model simulated equilibrium cooling response is also shown for reference as (-----). Lpg and ELP or Lpg[CPA] and ELp[CPA] that best fit the DSC water transport data of SVF, P0, P2 and P4 cells were used to generate the optimal cooling rates (data not shown). The optimal cooling rate, Bopt) was calculated using a recently developed Generic Optimal Cooling Rate Equation (GOCRE) [7] (data not shown). The GOCRE relates several cell level parameters (Lpg or Lpg[CPA]; ELP or ELp[CPA]; SA;WV) to Bopt as  Bopt 1009.5e 0.0546ELp Lpg  SA W V  .