Publications

Technologies to cryogenically preserve (a.k.a. cryopreserve) living tissue, cell lines and primary cells have matured greatly for both clinicians and researchers since their first demonstration in the 1950s and are widely used in storage and transport applications. Currently, however, there remains an absence of viable cryopreservation and thawing methods for bioengineered, three-dimensional (3D) cell models, including patients' samples. As a first step towards addressing this gap, we demonstrate a viable protocol for spheroid cryopreservation and survival based on a 3D carboxymethyl cellulose scaffold and precise conditions for freezing and thawing. The protocol is tested using hepatocytes, for which the scaffold provides both the 3D structure for cells to self-arrange into spheroids and to support cells during freezing for optimal post-thaw viability. Cell viability after thawing is improved compared to conventional pellet models where cells settle under gravity to form a pseudo-tissue before freezing. The technique may advance cryobiology and other applications that demand high-integrity transport of pre-assembled 3D models (from cell lines and in future cells from patients) between facilities, for example between medical practice, research and testing facilities.

JTD Keywords: 3d cell culture, biofabrication, biomaterials, carboxymethyl cellulose, cryopreservation, hepatocytes, 3d cell culture, Biofabrication, Biomaterials, Carboxymethyl cellulose, Cell culture techniques, three dimensional, Cell survival, Cryopreservation, Freezing, Hepatocytes, Humans, Prevention, Scaffolds, Spheroids

Cryopreservation methods for cell and tissue storage have beenaround since 1954, where thawed sperm samples were used for aninsemination. Since then, the technology has evolved for cliniciansand researchers to cryopreserve tissue, cell lines and primary cells.While cryopreserving tissues helps maintain their physiological in-tegrity for study it does not assure the viability of the cells afterthawing. Furthermore, the cells cryopreserved in suspension losetheir dimensional anchors, forcing them to change their morphology.To solve this issue, tissue engineering allows researchers to create 3Dculture models, such as organoids and bioprinted cell clusters, thatmimic the physiological characteristics of the cells in tissue anddisease. Although this culture methods present promising results,there is a lack of methodology to cryopreserve 3D cell models andpatients’ samples for storage and transport in a way where they re-main viable after thawing. We propose a protocol that uses a car-boxymethyl cellulose scaffold and precise freezing and thawingconditions for spheroid survival. The scaffold provides structure forthe hepatocytes to create spheroids on their own as well as supportthroughout the freezing and thawing processes for optimal cell via-bility post-thawing. Furthermore, this method will achieve highercell viability than transporting the cells as a cryopreserved pellet formodel assembling after thawing, allowing the cells to settle and forma tissue beforehand to improve viability after cryopreservation. Thistechnique constitutes a step forward for it will facilitate the transportof already assembled 3D models from cell lines or primary cells frompatients.

JTD Keywords: Cryopreservation

Visual impairments are a critical medical hurdle to be addressed in modern society. Müller glia (MG) have regenerative potential in the retina in lower vertebrates, but not in mammals. However, in mice, in vivo cell fusion between MG and adult stem cells forms hybrids that can partially regenerate ablated neurons.We used organotypic cultures of human retina and preparations of dissociated cells to test the hypothesis that cell fusion between human MG and adult stem cells can induce neuronal regeneration in human systems. Moreover, we established a microinjection system for transplanting human retinal organoids to demonstrate hybrid differentiation.We first found that cell fusion occurs between MG and adult stem cells, in organotypic cultures of human retina as well as in cell cultures. Next, we showed that the resulting hybrids can differentiate and acquire a proto-neural electrophysiology profile when the Wnt/beta-catenin pathway is activated in the adult stem cells prior fusion. Finally, we demonstrated the engraftment and differentiation of these hybrids into human retinal organoids.We show fusion between human MG and adult stem cells, and demonstrate that the resulting hybrid cells can differentiate towards neural fate in human model systems. Our results suggest that cell fusion-mediated therapy is a potential regenerative approach for treating human retinal dystrophies.This work was supported by La Caixa Health (HR17-00231), Velux Stiftung (976a) and the Ministerio de Ciencia e Innovación, (BFU2017-86760-P) (AEI/FEDER, UE), AGAUR (2017 SGR 689, 2017 SGR 926).Published by Elsevier B.V.

JTD Keywords: cell fusion, expression, fusion, ganglion-cells, in-vitro, mouse, müller glia, neural differentiation, organoids, regeneration, retina regeneration, stem cells, stromal cells, transplantation, 4',6 diamidino 2 phenylindole, 5' nucleotidase, Agarose, Alcohol, Arpe-19 cell line, Article, Beta catenin, Beta tubulin, Bone-marrow-cells, Bromophenol blue, Buffer, Calcium cell level, Calcium phosphate, Calretinin, Canonical wnt signaling, Cd34 antigen, Cell culture, Cell fusion, Cell viability, Coculture, Complementary dna, Confocal microscopy, Cornea transplantation, Cryopreservation, Cryoprotection, Crystal structure, Current clamp technique, Dimethyl sulfoxide, Dodecyl sulfate sodium, Edetic acid, Electrophysiology, Endoglin, Fetal bovine serum, Fibroblast growth factor 2, Flow cytometry, Fluorescence activated cell sorting, Fluorescence intensity, Glyceraldehyde 3 phosphate dehydrogenase, Glycerol, Glycine, Hoe 33342, Immunofluorescence, Immunohistochemistry, Incubation time, Interleukin 1beta, Lentivirus vector, Matrigel, Mercaptoethanol, Microinjection, Mueller cell, Müller glia, N methyl dextro aspartic acid, Nerve cell differentiation, Neural differentiation, Nitrogen, Nonhuman, Organoids, Paraffin, Paraffin embedding, Paraformaldehyde, Patch clamp technique, Penicillin derivative, Phenolsulfonphthalein, Phenotype, Phosphate buffered saline, Phosphoprotein phosphatase inhibitor, Polyacrylamide gel electrophoresis, Potassium chloride, Povidone iodine, Promoter region, Proteinase inhibitor, Real time polymerase chain reaction, Receptor type tyrosine protein phosphatase c, Restriction endonuclease, Retina, Retina dystrophy, Retina regeneration, Retinol, Rhodopsin, Rna extraction, Stem cell, Stem cells, Subcutaneous fat, Tunel assay, Visual impairment, Western blotting