Toward Better Cryopreservation: Xeno-Free, Reduced Toxicity Protocol

Biobanks play a critical role as functional components of research, meaning that cell cultivation and cryopreservation strategies need to be optimized. Two common components stand out as ripe for change:

• The use of animal serum, particularly fetal bovine serum (FBS), which is poorly defined and carries the risks of immunological reactions and transmission of zoonotic infection.

• The incorporation of toxic chemicals in the freezing media, particularly at high concentrations. One such chemical is the cryoprotective agent (CPA) dimethyl sulfoxide (DMSO).

In an effort to define an optimal xeno-free cultivation and cryopreservation protocol, Lauterboeck et al. (2016) recently analyzed three alternative cryoprotectants: methylcellulose, poloxamer-188 and α-tocopherol. The research team analyzed these cryoprotectants’ impact on viability and differentiation capacity when applied to multipotent stromal cells derived from the common marmoset (Callithrix jacchus).¹ They offer this data as a first report of cryopreservation using the CPA mixtures.

Analyzed CPAs

CPA	Components	Concentration (% v/v)
Control	DMEM, FBS, DMSO	75/22.5/5
MP	DMEM, DMSO, methylcellulose, poloxamer-188	96.4/2.5/0.1/1
MPT	DMEM, DMSO, methylcellulose, poloxamer-188, α-tocopherol	95.4/2.5/0.1/1/1

The research team first investigated the efficiency of cryoprotection for the CPA agents. They analyzed the CPA mixtures with varying concentrations of DMSO, different incubation times, and both with and without FBS. They found the best results (highest post-thaw cell survival) with either the MP or MPT combination at a 10-minute incubation period, without the addition of FBS and with DMSO reduced to 2.5%.

Further investigation revealed that a two-step cooling process (7.5°C/minute from 4°C to –30°C, then 3°C/minute from –30°C to –80°C) produced superior results in terms of cell survival and re-cultivation efficiency. The researchers used this protocol for subsequent experiments.

When it came to post-thaw metabolic activity, the team reported significant impairment of frozen cells for up to 48 hours when compared with non-frozen controls or 5% DMSO+FBS. By 96 hours post-thaw, all cryopreserved groups recovered metabolic activity, beginning with the MPT–FBS group at 72 hours.

The researchers observed no significant difference for doubling time. They reported 28±0.9 hours for non-frozen cells, 26±3.6 hours for 5% DMSO+FBS, 25±3 hours for MP-FBS, and 26±0.8 hours for MPT-FBS.

To investigate adipogenic differentiation potential, the team induced adipogenesis in frozen-thawed bone marrow multipotent stromal cells (BmMSC) and evaluated the differentiated cells after 20 days. They found that all BmMSCs formed adipocytes with typical fat vacuoles regardless of freezing conditions. They noted a significant decrease in oil-droplet formation for all frozen-thawed samples when compared with non-frozen cells, but saw no difference among the frozen-thawed groups. They also looked at two adipogenic markers (peroxisome proliferator-activated receptor gamma and adipsin) and reported no significant difference in expression among the MP and MPT groups when compared with controls.

The researchers also evaluated osteogenic differentiation potential by inducing osteogenesis in frozen-thawed BmMSCs and analyzing differentiated cells after 21 days. They report that all BmMSCs formed osteoblasts regardless of freezing conditions. They found no significant difference between frozen and non-frozen cells for measures of calcification of extracellular matrix or for expression of two osteogenic markers (osteocalcin and osteopontin) assessed via quantitative real-time polymerase chain reaction.

Overall, Lauterboeck et al. offer evidence that cryopreservation protocols that use alternative CPAs (methylcellulose, poloxamer-188 and α-tocopherol) and significantly lower DMSO concentrations (2.5% v/v) and that do not include serum produce excellent results in terms of both cell viability and differentiation capacity. This represents a critical first step toward the development of xeno-free cryopreservation with reduced toxicity from DMSO.

Reference

1. Lauterboeck, L., et al. (2016) “Xeno-free cryopreservation of bone marrow-derived multipotent stromal cells from Callithrix jacchus,” Biopreservation and Biobanking. doi: 10.1089/bio.2016.0038