First steps of an interdisciplinary approach towards miniaturised cryopreservation for cellular nanobiotechnology

Abstract

The only widely used and accepted method for long-term cell preservation is storage below -130 degrees C. The biosciences will make increasing use of preservation and place new demands on it. Currently, cells are frozen in volumes greater than 1 ml but the new cell and implantation therapies (particularly those using stem cells) will require accurately defined freezing and storage conditions for each single cell. Broadly-based, routine freezing of biological samples allows the advantage of retrospective analysis and the possibility of saving genetic rights. For such applications, one billion is a modest estimation for the number of samples. Current cryotechniques cannot handle so many samples in an efficient and economic way, and the need for new cryotechnology is evident. The interdisciplinary approach presented here should lead to a new sample storage and operating strategy that fulfils the needs mentioned above. Fundamental principles of this new kind of smart sample storage are: (i) miniaturisation; (ii) modularisation; (iii) informationsample integration, i.e. freezing memory chips with samples; and (iv) physical and logical access to samples and information without thawing the samples. In contrast to current sample systems, the prototyped family of intelligent cryosubstrates allows the recovery of single wells (parts) of the substrate without thawing the rest of the sample. The development of intelligent cryosubstrates is linked to developments in high throughput freezing, high packing density storage and minimisation of cytotoxic protective agents.

Cryopreservation in micro‐volumes: Impact upon caco‐2 colon adenocarcinoma cell proliferation and differentiation

“…These results suggest that the use of miniaturized cryosubstrates may be an intelligent solution for the storage of small volumes of a large number of samples, as already shown for the storage of the established L929 fibroblast cells (Zimmermann et al, 2004).…”

“…The cryoprotectants, Me 2 SO or glycerol (from 40% (v/v) stock) were added to give final concentrations of 10%, 5%, and 3% (v/v). They were either mixed with the cell suspension prior to filling micro-cryosubstrates and cryovials or added automatically to the individual wells by the micro-pipetting system Nano-Plotter NP1.2 (GeSiM mbH, Großerkmannsdorf, Germany) as described in previous work by Zimmermann et al (2004). The micro-cryosubstrates and cryovials were held at 48C for 20 min for cryoprotectant equilibration, frozen to À808C in an isopropanol-based freezing system, ''Mr.…”

“…The micro-cryosubstrates developed at the Fraunhofer Institute for Biomedicine Technique (IBMT) promise technical improvements due to the biophysical and logistical advantages of miniaturization, process automation, easy assess to aliquots of the sample and the potential for manipulating large sample numbers Zimmermann et al, 2004). Micro-cryosubstrates are made from high-density-polyethylene using injection-molding and contain 30 sample wells of 25 mL net volume in a plate of 4 cm  3 cm.…”

“…Micro-cryosubstrates are made from high-density-polyethylene using injection-molding and contain 30 sample wells of 25 mL net volume in a plate of 4 cm  3 cm. These micro-systems have smaller temperature gradients and non-homogeneities cryovials (Zimmermann et al, 2004). Furthermore, cryoprotectant can be quickly and precisely added to each well by a pipette robot system, giving homogeneous distribution and facilitating the handling of large sample numbers.…”

Nanopatterning Reveals an ECM Area Threshold for Focal Adhesion Assembly and Force Transmission that is regulated by Integrin Activation and Cytoskeleton Tension

“…2A). In some instances, integrin staining was present over non-adhesive areas spanning adhesive nanoislands corresponding to 'cell bridging' (Lehnert et al, 2004;Zimmermann et al, 2004;Rossier et al, 2010). We do not expect that integrins over the non-adhesive areas contribute significantly to adhesive force since there is no FN underneath them (Fig.…”