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The aim of this study was to compare concentrations of endogenous N-acylethanolamine (NAE) lipid mediators—palmitoylethanolamide (PEA), oleoylethanolamide (OEA), and anandamide (AEA)—in fresh, decontaminated, cryopreserved, and freeze-dried amniotic membrane (AM) allografts, thereby determining whether AM’s analgesic and anti-inflammatory efficiency related to NAEs persists during storage. The concentrations of NAEs were measured using ultra-high-performance liquid chromatography–tandem mass spectrometry. Indirect fluorescent immunohistochemistry was used to detect the PEA PPAR-α receptor. The concentrations of PEA, OEA, and AEA were significantly higher after decontamination. A significant decrease was found in cryopreserved AM compared to decontaminated tissue for PEA but not for OEA and AEA. However, significantly higher values for all NAEs were detected in cryopreserved samples compared to fresh tissue before decontamination. The freeze-dried AM had similar values to decontaminated AM with no statistically significant difference. The nuclear staining of the PPAR-α receptor was clearly visible in all specimens. The stability of NAEs in AM after cryopreservation was demonstrated under tissue bank storage conditions. However, a significant decrease, but still higher concentration of PEA compared to fresh not decontaminated tissue, was found in cryopreserved, but not freeze-dried, AM. Results indicate that NAEs persist during storage in levels sufficient for the analgesic and anti-inflammatory effects. This means that cryopreserved AM allografts released for transplant purposes before the expected expiration (usually 3–5 years) will still show a strong analgesic effect. The same situation was confirmed for AM lyophilized after one year of storage. This work thus contributed to the clarification of the analgesic effect of NAEs in AM allografts.

Abstract Destruction or dysfunction of limbal epithelial stem cells (LESCs) leads to unilateral or bilateral limbal stem cell deficiency (LSCD). Fifteen years have passed since the first transplantation of ex vivo cultivated oral mucosal epithelial cells (COMET) in humans in 2004, which represents the first use of a cultured non-limbal autologous cell type to treat bilateral LSCD. This review summarizes clinical outcomes from COMET studies published from 2004 to 2019 and reviews results with emphasis on the culture methods by which grafted cell sheets were prepared.

Transplantation of novel tissue-engineered products using cultured epithelial cells is gaining significant interest. While such treatments can readily be provided at centralized medical centers, delivery to patients at geographically remote locations requires the establishment of suitable storage protocols. One important aspect of storage technology is temperature. This paper reviews storage temperature for above-freezing point storage of human epithelial cells for regenerative medicine purposes. The literature search uncovered publications on epidermal cells, retinal pigment epithelial cells, conjunctival epithelial cells, corneal/limbal epithelial cells, oral keratinocytes, and seminiferous epithelial cells. The following general patterns were noted: (1) Several studies across different cell types inclined toward 4 and 16°C being suitable short-term storage temperatures. Correspondingly, almost all studies investigating 37°C concluded that this storage temperature was suboptimal. (2) Cell death typically escalates rapidly following 7–10 days of storage. (3) The importance of the type of storage medium and its composition was highlighted by some of the studies; however, the relative importance of storage medium vs. storage temperature has not been investigated systematically. Although a direct comparison between the included investigations is not reasonable due to differences in cell types, storage media, and storage duration, this review provides an overview, summarizing the work carried out on each cell type during the past two decades.

Transplantation of cultured epidermal cell sheets (CES) can be life-saving for patients with large area burns. CES have also been successfully used to regenerate eye and urethral epithelia in animal models. Short-term storage aims to extend the transplantation window, offers flexibility in timing surgery and allows testing of CES quality, phenotype and sterility. This study investigated extended CES storage and explored the effect of additional re-incubation recovery time following storage. The proliferative quality of stored confluent versus pre-confluent CES was also investigated using functional testing. CES were stored at 12°C and results compared to non-stored control CES. Investigation of timepoints during 15 days storage revealed that viability began to deteriorate by day 11 and was associated with increased lactate in the storage medium. The percentage of apoptotic cells also significantly increased by day 11. Flow cytometry analysis of integrin β1 expression and cell size indicated best retention of stem cells at 7 days of storage. Functional testing of pre-confluent and confluent cells following 7 days storage showed that pre-confluent cells responded well to 1-day re-incubation after storage; they became highly prolific, increasing in number by ~67%. Conversely, proliferation in stored confluent cells declined by ~50% with 1-day re-incubation. Pre-confluent stored CES also had far superior stem cell colony forming efficiency (CFE) performance compared to the confluent group. Re-incubation improved CFE in both groups, but the pre-confluent group again out-performed the confluent group with significantly more colonies. In conclusion, a maximum storage period of 7 days is recommended. Use of pre-confluent cells and one day recovery incubation greatly improves viability, colony-forming ability and proliferation of cells stored for 7 days at 12°C. Thus, these recommendations should be considered under culture and storage of high-quality CES for clinical use.

We previously demonstrated that the silk protein sericin promotes pigmentation of retinal pigment epithelium (RPE) by activating the NF-κB pathway. Among numerous agents, NF-κB can be activated by hydrogen peroxide. In the present study, we explored possible associations between reactive oxygen species and sericin-induced melanogenesis in RPE. The proteome of human fetal RPE cultured for seven days with or without 1% sericin was analyzed using ingenuity pathway analysis (IPA). The proteomic data was verified by immunofluorescence and immunoblotting. Light microscopy and scanning electron microscopy were used to assess morphology. Dihydroethidium (DHE) and dihydrorhodamine (DHR) assays were used to measure superoxide and hydrogen peroxide species. Expression levels of proteins related to inflammation, differentiation, cell survival and cell adhesion were higher in cells cultured in Dulbecco’s Modified Eagle Medium (DMEM) with 1% sericin, whereas cells cultured in DMEM alone showed higher expression levels of proteins associated with Bruch’s membrane and cytoskeleton. Despite upregulation of inflammatory proteins, sericin co-cultured RPE yielded significantly higher cell viability compared to cells cultured without sericin. Addition of sericin to culture media significantly increased hydrogen peroxide-levels without significantly affecting superoxide-levels. We suggest that sericin-induced melanogenesis in cultured RPE is associated with elevated levels of superoxide dismutase, hydrogen peroxide and inflammatory proteins.

There is a need to optimize storage conditions to preserve cell characteristics during transport of cultured cell sheets from specialized culture units to distant hospitals. In this study, we aimed to explore a method to identify additives that diminish the decrease in the viability of stored undifferentiated epidermal cells using multifactorial design and an automated screening procedure. The cultured cells were stored for 7–11 days at 12°C in media supplemented with various additives. Effects were evaluated by calcein staining of live cells as well as morphology. Twenty-six additives were tested using (1) a two-level factorial design in which 10 additives were added or omitted in 64 different combinations and (2) a mixture design with 5 additives at 5 different concentrations in a total of 64 different mixtures. Automated microscopy and cell counting with Fiji enabled efficient processing of data. Significant regression models were identified by Design-Expert software. A calculated maximum increase of live cells to 37 ± 6% was achieved upon storage of cell sheets for 11 days in the presence of 6% glycerol. The beneficial effect of glycerol was shown for epidermal cell sheets from three different donors in two different storage media and with two different factorial designs. We have thus developed a high throughput screening system enabling robust assessment of live cells and identified glycerol as a beneficial additive that has a positive effect on epidermal cell sheet upon storage at 12°C. We believe this method could be of use in other cell culture optimization strategies where a large number of conditions are compared for their effect on cell viability or other quantifiable dependent variables.

Hyaluronan (HA), a major component of the extracellular matrix, plays a key role in cell proliferation, growth, survival, polarization and differentiation. We investigated the optimization of a HA hydrogel scaffold for culture of human oral mucosal epithelial cells (OMECs) for potential application in limbal stem cell therapy. The effect of the optimized scaffold on OMEC cell sheet morphology, cell metabolic activity and expression of genes associated with stemness, adherence and cell damage was studied. The results indicate that HA hydrogels crosslinked with polyethylene glycol diacrylate (PEGDA) failed to support OMEC attachment and growth. However, HA hydrogel scaffolds dried for three days and coated with 1 mg/mL collagen IV produced a full OMEC sheet. Cell morphology was comparable to control after three weeks culture, maintaining 76% metabolic activity. Of apoptosis-related genes, the pro-apoptotic markers CASP3 and BAX2 were upregulated and downregulated, respectively, compared to control whereas the anti-apoptotic marker BCL2 was downregulated. The expression level of stemness genes ΔNp63α and ABCG2 was significantly higher than control. Genes associated with improved scar-less wound healing (integrin-αV) and protection of the ocular surface (cadherin-1) had ~3-fold increased expression. These data suggest that our optimized HA-hydrogel scaffold could enhance culture of OMEC cell sheets for use in ocular reconstruction.

Hyaluronan (HA), also termed hyaluronic acid or hyaluronate, is a major component of the extracellular matrix. This non-sulfated glycosaminoglycan plays a key role in cell proliferation, growth, survival, polarization, and differentiation. The diverse biological roles of HA are linked to the combination of HA’s physicochemical properties and HA-binding proteins. These unique characteristics have encouraged the application of HA-based hydrogel scaffolds for stem cell-based therapy, a successful method in the treatment of limbal stem cell deficiency (LSCD). This condition occurs following direct damage to limbal stem cells and/or changes in the limbal stem cell niche microenvironment due to intrinsic and extrinsic insults. This paper reviews the physical properties, synthesis, and degradation of HA. In addition, the interaction of HA with other extracellular matrix (ECM) components and receptor proteins are discussed. Finally, studies employing HA-based hydrogel scaffolds in the treatment of LSCD are reviewed.