Cryopreservation effects on intermediary metabolism in a pancreatic substitute: a (13)C nuclear magnetic resonance study.

Tissue Eng Part A. 2012 Nov;18(21-22):2323-31

Authors: Ahmad HF, Simpson NE, Lawson AN, Sambanis A

Abstract
Cryopreservation is important for clinical translation of tissue-engineered constructs. With respect to a pancreatic substitute, encapsulated islets or beta cells have been widely studied for the treatment of insulin-dependent diabetes mellitus. Besides cell viability loss, cryopreservation may affect the function of the remaining viable cells in a pancreatic substitute by altering fundamental processes in glucose-stimulated insulin secretion, such as pathways associated with intermediary metabolism, potentially leading to insulin-secretion defects. In this study, we used (13)C nuclear magnetic resonance (NMR) spectroscopy and isotopomer analysis to determine the effects of conventional freezing and ice-free cryopreservation (vitrification) on carbon flow through tricarboxylic acid (TCA) cycle-associated pathways in encapsulated murine insulinoma βTC-tet cells; the secretory function of the encapsulated cells postpreservation was also evaluated. Specifically, calcium alginate-encapsulated βTC-tet cells were frozen or vitrified with a cryoprotectant cocktail. Beads were warmed and (13)C labeling and extraction were performed. Insulin secretion rates were determined during basal and labeling periods and during small-scale glucose stimulation and K(+)-induced depolarization. Relative metabolic fluxes were determined from (13)C NMR spectra using a modified single pyruvate pool model with the tcaCALC modeling program. Treatments were compared with nonpreserved controls. Results showed that relative carbon flow through TCA-cycle-associated pathways was not affected by conventional freezing or vitrification. However, vitrification, but not freezing, led to impaired insulin secretion on a per viable cell basis. The reduced secretion from the Vitrified group occurred irrespective of scale and was present whether secretion was stimulated by glucose or K(+)-induced depolarization, indicating that it might be due to a defect in late-stage secretion events.

PMID: 22697373 [PubMed - indexed for MEDLINE]

Cryopreservation Effects on Recombinant Myoblasts Encapsulated in Adhesive Alginate Hydrogels.

Acta Biomater. 2013 Mar 11;

Authors: Ahmad HF, Sambanis A

Abstract
Cell encapsulation in hydrogels is widely used in tissue engineering applications, including encapsulation of islets or other insulin-secreting cells in pancreatic substitutes. Use of adhesive, bio-functionalized hydrogels is receiving increasing attention, as cell-matrix interactions in 3-D can be important for various cell processes. With pancreatic substitutes, studies have indicated benefits of 3-D adhesion on the viability and/or function of insulin-secreting cells. As long-term storage of microencapsulated cells is critical for their clinical translation, cryopreservation of cells in hydrogels is actively being investigated. Previous studies have examined the cryopreservation response of cells encapsulated in non-adhesive hydrogels using conventional freezing and/or vitrification (ice-free cryopreservation), however, none have systematically compared the two cryopreservation methods with cells encapsulated within an adhesive 3-D environment. The latter would be significant, as evidence suggests adhesion influences cellular response to cryopreservation. Thus, the objective of this study was to determine the response to conventional freezing and vitrification of insulin-secreting cells encapsulated in an adhesive biomimetic hydrogel. Recombinant insulin-secreting C2C12 myoblasts were encapsulated in oxidized RGD-alginate and cultured 1 or 4 days post-encapsulation, cryopreserved, and assessed up to 3 days post-warming for metabolic activity and insulin secretion, and one day post-warming for cell morphology. Besides certain transient differences of the vitrified group relative to the Fresh control, both conventional freezing and vitrification maintained metabolism, secretion and morphology of the recombinant C2C12 cells. Thus, due to a simpler procedure and slightly superior results, conventional freezing is recommended over vitrification for the cryopreservation of C2C12 cells in oxidized RGD-modified alginate.

PMID: 23499987 [PubMed - as supplied by publisher]

Dual factor delivery of CXCL12 and Exendin-4 for improved survival and function of encapsulated beta cells under hypoxic conditions.

Biotechnol Bioeng. 2013 Feb 21;

Authors: Duncanson S, Sambanis A

Abstract
A bioartifical pancreas (BAP) remains a promising approach for treating insulin-dependent diabetes. Several obstacles to the clinical implementation of a BAP remain, including hypoxia following implantation. Within native pancreatic islets, CXCL12 and glucagon-like peptide-1 (GLP-1) act in a paracrine fashion to promote the survival, function, and proliferation of β-cells. This work sought to investigate if the presentation of CXCL12 and delivery of a GLP-1 receptor analog, Exendin-4 (Ex-4), alone and in combination, conferred pro-survival and insulinotropic effects on an encapsulated β-cell line, βTC-tet, cultured under hypoxic conditions of 7.6 mmHg O(2) . Our findings indicate that presentation of CXCL12 in the encapsulation matrix completely abrogated apoptosis under hypoxic conditions. Delivery of Ex-4 increased insulin secretion rate under both normoxic and hypoxic conditions, and additionally reduced apoptosis under hypoxic conditions. Furthermore, presentation of CXCL12 combined with Ex-4 delivery significantly increased insulin secretion rate under hypoxic conditions compared to delivery of Ex-4 alone. These findings demonstrate that the presentation of CXCL12 combined with the delivery of Ex-4 may constitute a promising strategy for supporting β-cell function and survival following transplantation. Biotechnol. Bioeng. © 2013 Wiley Periodicals, Inc.

PMID: 23436382 [PubMed - as supplied by publisher]

Alginate microencapsulation of human mesenchymal stem cells as a strategy to enhance paracrine-mediated vascular recovery after hindlimb ischaemia.

J Tissue Eng Regen Med. 2012 Dec 21;

Authors: Landázuri N, Levit RD, Joseph G, Ortega-Legaspi JM, Flores CA, Weiss D, Sambanis A, Weber CJ, Safley SA, Taylor WR

Abstract
Stem cell-based therapies hold great promise as a clinically viable approach for vascular regeneration. Preclinical studies have been very encouraging and early clinical trials have suggested favourable outcomes. However, significant challenges remain in terms of optimizing cell retention and maintenance of the paracrine effects of implanted cells. To address these issues, we have proposed the use of a cellular encapsulation approach to enhance vascular regeneration. We contained human mesenchymal stem cells (hMSCs) in biocompatible alginate microcapsules for therapeutic treatment in the setting of murine hindlimb ischaemia. This approach supported the paracrine pro-angiogenic activity of hMSCs, prevented incorporation of hMSCs into the host tissue and markedly enhanced their therapeutic effect. While injection of non-encapsulated hMSCs resulted in a 22 ± 10% increase in vascular density and no increase in perfusion, treatment with encapsulated hMSCs resulted in a 70 ± 8% increase in vascular density and 21 ± 7% increase in perfusion. The described cellular encapsulation strategy may help to better define the mechanisms responsible for the beneficial effects of cell-based therapies and provide a therapeutic strategy for inducing vascular growth in the adult. As hMSCs are relatively easy to isolate from patients, and alginate is biocompatible and already used in clinical applications, therapeutic cell encapsulation for vascular repair represents a highly translatable platform for cell-based therapy in humans. Copyright © 2012 John Wiley & Sons, Ltd.

PMID: 23281223 [PubMed - as supplied by publisher]