RNA METABOLISM IN HELA CELLS AT REDUCED TEMPERATURE : I. Modified Processing of 45S RNA

Incubation of HeLa cells at suboptimal temperature has been used to study the synthesis of 45S ribosomal RNA precursor and the individual steps of the subsequent processing to 28S RNA. Below 20°C no detectable 45S RNA is formed. The processing of 45S RNA to 32S RNA ceases around 15°C, and the processing of 32S RNA to 28S RNA is inhibited near 25°C. Prolonged incubation at reduced temperature results in further modification of the processing, resulting in the apparent accumulation of 41S RNA. The products of these reactions at reduced temperature appear normal in that the ribosomal RNA made at 27°C can be isolated from functional polyribosomes in the cytoplasm after a short incubation at 37°C.     

A STUDY OF THE PENETRATION OF MAMMALIAN CELLS BY DEOXYRIBONUCLEIC ACIDS

Tritium-labeled deoxyribonucleic acid (DNA) from pneumococci and from human leukocytes was added to growing cultures of HeLa cells at 37°C. Autoradiography revealed an extensive localization of tritium in the nuclear regions. The label could not be removed by treatment with ribonuclease or dilute perchloric acid, but quantitative removal from the cells could be effected with deoxyribonuclease. Chemical and radioactivity determinations on nucleic acids isolated from the exposed HeLa cells revealed the presence of tritium in all 4 DNA bases. About 12 µg. of tritiated DNA was recovered from 6 x 10⁶ HeLa cells which had been exposed for 24 hours to 240 µg. of the human DNA. From this, it is concluded that the amount of DNA, or its degradation products, taken up by the cells was equivalent to at least 10 per cent of the normal HeLa cell complement.     

Protein cofactors and substrate influence Mg2+-dependent structural changes in the catalytic RNA of archaeal RNase P

The ribonucleoprotein (RNP) form of archaeal RNase P comprises one catalytic RNA and five protein cofactors. To catalyze Mg²⁺-dependent cleavage of the 5′ leader from pre-tRNAs, the catalytic (C) and specificity (S) domains of the RNase P RNA (RPR) cooperate to recognize different parts of the pre-tRNA. While ∼250–500 mM Mg²⁺ renders the archaeal RPR active without RNase P proteins (RPPs), addition of all RPPs lowers the Mg²⁺ requirement to ∼10–20 mM and improves the rate and fidelity of cleavage. To understand the Mg²⁺- and RPP-dependent structural changes that increase activity, we used pre-tRNA cleavage and ensemble FRET assays to characterize inter-domain interactions in Pyrococcus furiosus (Pfu) RPR, either alone or with RPPs ± pre-tRNA. Following splint ligation to doubly label the RPR (Cy3-RPR_{C domain} and Cy5-RPR_{S domain}), we used native mass spectrometry to verify the final product. We found that FRET correlates closely with activity, the Pfu RPR and RNase P holoenzyme (RPR + 5 RPPs) traverse different Mg²⁺-dependent paths to converge on similar functional states, and binding of the pre-tRNA by the holoenzyme influences Mg²⁺ cooperativity. Our findings highlight how Mg²⁺ and proteins in multi-subunit RNPs together favor RNA conformations in a dynamic ensemble for functional gains.     

Effect of Storage Temperature on Cultured Epidermal Cell Sheets Stored in Xenobiotic-Free Medium

Cultured epidermal cell sheets (CECS) are used in regenerative medicine in patients with burns, and have potential to treat limbal stem cell deficiency (LSCD), as demonstrated in animal models. Despite widespread use, short-term storage options for CECS are limited. Advantages of storage include: flexibility in scheduling surgery, reserve sheets for repeat operations, more opportunity for quality control, and improved transportation to allow wider distribution. Studies on storage of CECS have thus far focused on cryopreservation, whereas refrigeration is a convenient method commonly used for whole skin graft storage in burns clinics. It has been shown that preservation of viable cells using these methods is variable. This study evaluated the effect of different temperatures spanning 4°C to 37°C, on the cell viability, morphology, proliferation and metabolic status of CECS stored over a two week period in a xenobiotic–free system. Compared to non-stored control, best cell viability was obtained at 24°C (95.2±9.9%); reduced cell viability, at approximately 60%, was demonstrated at several of the temperatures (12°C, 28°C, 32°C and 37°C). Metabolic activity was significantly higher between 24°C and 37°C, where glucose, lactate, lactate/glucose ratios, and oxygen tension indicated increased activation of the glycolytic pathway under aerobic conditions. Preservation of morphology as shown by phase contrast and scanning electron micrographs was best at 12°C and 16°C. PCNA immunocytochemistry indicated that only 12°C and 20°C allowed maintenance of proliferative function at a similar level to non-stored control. In conclusion, results indicate that 12°C and 24°C merit further investigation as the prospective optimum temperature for short-term storage of cultured epidermal cell sheets.     

Relationship between Freezing Tolerance of Root-Tip Cells and Cold Stability of Microtubules in Rye (Secale cereale L. cv Puma)

The response of cortical microtubules to low temperature and freezing was assessed for root tips of cold-acclimated and non-acclimated winter rye (Secale cereale L. cv Puma) seedlings using indirect immunofluorescence microscopy with antitubulin antibodies. Roots cooled to 0 or −3°C were fixed for immunofluorescence microscopy at these temperatures or after an additional hour at 4°C. Typical arrays of cortical microtubules were present in root-tip cells of seedlings exposed to the cold-acclimation treatment of 4°C for 2 days. Microtubules in these cold-acclimated cells were more easily depolymerized by a 0°C treatment than microtubules in root-tip cells of nonacclimated, 22°C-grown seedlings. Microtubules were still present in some cells of both nonacclimated and cold-acclimated roots at 0 and −3°C; however, the number of microtubules in these cells was lower than in controls. Microtubules remaining during the −3°C freeze were shorter than microtubules in unfrozen control cells. Repolymerization of microtubules after both the 0 and −3°C treatments occurred within 1 h. Root tips of nonacclimated seedlings had an LT-50 of −9°C. Cold acclimation lowered this value to −14°C. Treatment of 22°C-grown seedlings for 24 h with the microtubule-stabilizing drug taxol caused a decrease in the freezing tolerance of root tips, indicated by a LT-50 of −3°C. Treatment with D-secotaxol, an analog of taxol that was less effective in stabilizing microtubules, did not alter the freezing tolerance. We interpret these data to indicate that a degree of depolymerization of microtubules is necessary for realization of maximum freezing tolerance in root-tip cells of rye.     

A COMPARISON BETWEEN HETEROGENEOUS NUCLEAR RNA AND POLYSOMAL MESSENGER RNA IN HELA CELLS BY RNA-DNA HYBRIDIZATION

Heterogeneous nuclear RNA (HnRNA) and mRNA from cytoplasmic polyribosomes of HeLa cells have been compared by RNA-DNA hybridization tests. 1 µg of HeLa cell DNA binds 0.05–0.10 µg of either HnRNA or mRNA. In addition, HeLa DNA that is preexposed to unlabeled HnRNA was found to have a reduced capacity to bind either HnRNA or mRNA. The results are compatible with considerable sequence similarity in the two types of RNA but, as is discussed, firm conclusions are precluded by imperfections of the hybridization reaction as presently employed.     

Effects of Temperature on Resistance in Phaseolus vulgaris Genotypes and on Development of Meloidogyne Species

Phaseolus vulgaris lines with heat-stable resistance to Meloidogyne spp. may be needed to manage root-knot nematodes in tropical regions. Resistance expression before and during the process of nematode penetration and development in resistant genotypes were studied at pre- and postinoculation temperatures of 24 °C and 24 °C, 24 °C and 28 °C, 28 °C and 24 °C, and 28 °C and 28 °C. Resistance was effective at all temperature regimes examined, with fewer nematodes in roots of a resistant line compared with a susceptible line. Preinoculation temperature did not modify resistance expression to later infections by root-knot nematodes. However, postinoculation temperatures affected development of Meloidogyne spp. in both the resistant and susceptible bean lines tested. The more rapid development of nematodes to adults at the higher postinoculation temperature of 28 °C in both bean lines suggests direct temperature effects on nematode development instead of on resistance expression of either of two gene systems. Also, resistance was stable at 30 °C and 32 °C.     

Enhancing Protein Expression in HEK-293 Cells by Lowering Culture Temperature

Animal cells and cell lines, such as HEK-293 cells, are commonly cultured at 37°C. These cells are often used to express recombinant proteins. Having a higher expression level or a higher protein yield is generally desirable. As we demonstrate in this study, dropping culture temperature to 33°C, but not lower, 24 hours after transient transfection in HEK-293S cells will give rise to ~1.5-fold higher expression of green fluorescent protein (GFP) and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors. By following the time course of the GFP-expressing cells growing at 37°C and 33°C from 24 hours after transfection (including 19 hours recovery at 37°C in the normal growth medium), we found that a mild hypothermia (i.e., 33°C) reduces the growth rate of HEK-293S cells, while increasing cellular productivity of recombinant proteins. As a result, green cells remain undivided in a longer period of time. Not surprisingly, the property of a recombinant protein expressed in the cells grown at 33°C is unaffected, as shown by the use of AMPA receptors. We further demonstrate with the use of PC12 cells that this method may be especially useful when a recombinant protein is difficult to express using a chemical-based, transient transfection method.     

Stability of hematological analytes during 48 hours storage at three temperatures using Cell-Dyn hematology analyzer

BackgroundThe complete blood count (CBC) with differential leukocyte count (DLC) is one of the most common tests requested by physicians. The results of this test are affected by storage temperature and time of incubation. This study was designed to evaluate the stability of hematologic parameters in blood specimens stored for 48 h at three temperatures.MethodsK2-EDTA - blood was collected from 22 healthy adults. The CBC was performed using a hematology analyser immediately; 0 time point and at 4, 8, 12, 16, 20, 24, and 48 h after storage at 4 °C, 10 °C or 23 °C. Changes in values of CBC parameters from the 0 time point were determined and reported as % of the initial value.ResultsRed blood cells, platelet, hemoglobin, and mean corpuscular hemoglobin were found stable during 48 h storage at 4 °C, 10 °C or 23 °C. Hematocrite and mean corpuscular volume increased, while white blood cells decreased at 48 h when stored at 23 °C. Lymphocytes, neutrophils, eosinophils, and basophils showed significant differences after 12 h of storage at 23 °C.ConclusionsRed blood cells, platelet, hemoglobin, and mean corpuscular hemoglobin are the only suitable parameters without refrigeration during 24 h storage. When CBC and DLC are performed, 4 °C can be recommended as the most suitable storage temperature for 12 h storage.     

Low concentrations of doxycycline attenuates FasL-induced apoptosis in HeLa cells

Background

Doxycycline (DC) has been shown to possess non-antibiotic properties including Fas/Fas Ligand (FasL)-mediated apoptosis against several tumor types in the concentration range of 10–40 µg/mL. However, the effect of DC in apoptotic signaling at much low concentrations was not studied.

Methods

The present study investigated the attenuation effect of low dose of DC on FasL-induced apoptosis in HeLa cell by the methods of MTT assay, fluorescence microscopy, DNA fragmentation, flow cytometry analysis, and western blotting.

Results and conclusion

In the present findings we showed that low concentration of DC (<2.0 µg/mL) exhibited protective effects against FasL-induced apoptosis in HeLa cells. FasL treatment to HeLa cells resulted in a concentration-dependent induction of cell death, and treatment with low concentrations of DC (0.1–2 µg/mL) significantly (p < 0.001) attenuated the FasL-induced cell death as measured by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Further, the FasL-induced apoptotic features in HeLa cells, such as morphological changes, DNA fragmentation and cell cycle arrest was also inhibited by DC (0.5 µg/mL). Tetracycline and minocycline also showed similar anti-apoptotic effects but were not significant when compared to DC, tested at same concentrations. Further, DC (0.01–16 µg/mL) did not influence the hydrogen peroxide- or cisplatin-induced intrinsic apoptotic pathway in HeLa cells. Protein analysis using Western blotting confirmed that FasL-induced cleavage/activation of caspase-8 and caspase-3, were inhibited by DC treatment at low concentration (0.5 µg/mL). Considering the overall data, we report for the first time that DC exhibited anti-apoptotic effects at low concentrations in HeLa cells by inhibition of caspase activation via FasL-induced extrinsic pathway.

Electronic supplementary material

The online version of this article (doi:10.1186/s40659-015-0025-8) contains supplementary material, which is available to authorized users.     

EXPERIMENTS ON TOLERATION OF TEMPERATURE BY DROSOPHILA

1. Most wild stocks of Drosophila melanogaster can be bred indefinitely on banana agar at a temperature of 31°C. There is no relation between the geographical origin of these stocks and their ability to tolerate this temperature. 2. A single wild stock has been found which will breed for only one generation at temperatures above 29°C. The offspring hatched at 31°C. will breed normally at 24°C. This difference from other wild stocks is apparently genetic, but its genetic basis has not yet been worked out. 3. The mutant stocks of D. melanogaster tested by us will breed for only one generation at 31°C. and their offspring at this temperature are also fertile at 24°C. This condition is apparently a physiological effect of the presence of any of the mutant genes in a homozygous condition. 4. Similar tests indicate that wild stocks of D. virilis and Chymomyza procnemis will breed at 31°C., while D. simulans, D. immigrans, and D. funebris will not. The last two species are northern forms not commonly found in the tropics. 5. Both male and female flies from mutant stocks hatched at 31°C. produce offspring at this temperature if mated to flies hatched at 24°C. Their germ cells are therefore capable of development, and the cause of their failure to develop at 31°C. when inbred must lie either in the failure of the germ cells to reach each other or in the fertilization process itself.     

MORPHOLOGY OF RIGOR-SHORTENED BOVINE MUSCLE AND THE EFFECT OF TRYPSIN ON PRE- AND POSTRIGOR MYOFIBRILS

Bovine semitendinosus muscles were sampled immediately after death, after 24 hr postmortem with storage at 2°, 16°, or 37°C, and after 312 hr postmortem with storage at 2° and 16°C. A biopsy technique was used to prevent shortening during glutaraldehyde fixation. Postfixation in osmium tetroxide was followed by embedding in an Epon-Araldite mixture. Bovine muscle was supercontracted after 24 hr storage at 27deg; but was only slightly contracted after storage at 16° for 24 hr. Muscle held at 37° for 24 hr was slightly less supercontracted than the 2° muscle. Striking similarities existed between muscles stored at 16° and at 2°C for 312 hr. Both were slightly shortened with narrowed I bands and an area of increased density, probably due to overlap of thin filaments in the middle of the A band. Postmortem shortening was accompanied by banding-pattern changes similar to those predicted for contracting muscle by Huxley and Hanson's sliding filament model. Treatment of myofibrils with 0.05% trypsin resulted in a rapid loss of Z lines and, in supercontracted myofibrils, caused a return of the banding pattern of resting muscle.     

Effect of Temperature Oscillation on Insect Cell Growth and Baculovirus Replication

Temperature oscillation can enhance cell viability of sf9 insect cells and baculovirus production of occlusion bodies (OB) and extracellular virus (ECV) compared with constant temperature in stationary culture and suspension culture. The optimal oscillation range was 24 to 28°C. At this temperature oscillation, the viability of uninfected and infected sf9 cells can be maintained much longer than at 28°C. Although the rate of virus infection was a little low at 24 to 28°C, the final cell infectivity was similar to that at a constant temperature of 28°C. The production of OB was increased from 13.4 to 17.4/cell in stationary culture and from 13.9/cell to 18.1/cell in suspension culture. The titer of ECV was increased from 87 to 114 PFU/cell in stationary culture and from 79 to 114 PFU/cell in suspension culture.     

Impact of Daily Thermocycles on Hatching Rhythms,Larval Performance and Sex Differentiation of Zebrafish

In the wild, water temperature cycles daily: it warms up after sunrise, and cools rapidly after sunset. Surprisingly, the impact of such daily thermocycles during the early development of fish remains neglected. We investigated the influence of constant vs daily thermocycles in zebrafish, from embryo development to sexual differentiation, by applying four temperature regimens: two constant (24°C and 28°C) and two daily thermocycles: 28:24°C, TC (thermophase coinciding with daytime, and cryophase coinciding with night-time) and 24:28°C, CT (opposite to TC) in a 12:12 h light:dark cycle (LD). Embryo development was temperature-dependent but enhanced at 28°C and TC. Hatching rhythms were diurnal (around 4 h after lights on), but temperature- and cycle-sensitive, since hatching occurred sooner at 28°C (48 hours post fertilization; hpf) while it was delayed at 24°C (96 hpf). Under TC, hatching occurred at 72 hpf, while under CT hatching displayed two peaks (at 70 hpf and 94 hpf). In constant light (LL) or darkness (DD), hatching rhythms persisted with tau close to 24 h, suggesting a clock-controlled “gating” mechanism. Under 28°C or TC, larvae showed the best performance (high growth and survival, and low malformations). The sex ratio was strongly influenced by temperature, as the proportion of females was higher in CT and TC (79 and 83% respectively), contrasting with 28°C and 24°C, which led to more males (83 and 76%). Ovarian aromatase (cyp19a) expression in females was highest in TC and CT (6.5 and 4.6 fold higher than at 28°C, respectively); while anti-müllerian hormone (amh) expression in males increased in testis at 24°C (3.6 fold higher compared to TC) and particularly at 28°C (14.3 fold increase). Taken together, these findings highlight the key role of environmental cycles during early development, which shaped the daily rhythms in fish embryo and larvae, and ultimately influenced sex differentiation.     

Indirect Measurement of the Lag Time Distribution of Single Cells of Listeria innocua in Food

The distribution of log counts at a given time during the exponential growth phase of Listeria innocua measured in food samples inoculated with one cell each was applied to estimate the distribution of the single-cell lag times. Three replicate experiments in broth showed that the distribution of the log counts is a linear mapping of the distribution of the detection times measured by optical density. The detection time distribution reflects the lag time distribution but is shifted in time. The log count distribution was applied to estimate the distributions of the lag times in a liquid dairy product and in liver paté after different heat treatments. Two batches of ca. 100 samples of the dairy product were inoculated and heated at 55°C for 45 min or at 62°C for 2 min, and an unheated batch was incubated at 4°C. The final concentration of surviving bacteria was ca. 1 cell per sample. The unheated cells showed the shortest lag times with the smallest variance. The mean and the variance of the lag times of the surviving cells at 62°C were greater than those of the cells treated at 55°C. Three batches of paté samples were heated at 55°C for 25 min, 62°C for 81 s, or 65°C for 20 s. A control batch was inoculated but not heated. All paté samples were incubated at 15°C. The distribution of the lag times of the cells heated at 55°C was not significantly different from that of the unheated cells. However, at the higher temperatures, 62°C and 65°C, the lag duration was longer and its variance greater.     

Effect of Chilling Temperatures upon Cell Cultures of Tomato

The effect of chilling temperatures upon cell cultures of tomato (Lycopersicon esculentum Mill cv `VF36,' and cv `VFNT Cherry,' and L. hirsutum Humb. & Bonpl.) was tested. Doubling times for L. esculentum were 2 to 3 days at 28°C, and 3 to 8 days at 12°C. No growth was observed at 8°C, indicating an abrupt limit to growth between 8 and 12°C. Fluorescein diacetate staining indicated that 80 to 90% of the cells were alive when cells were maintained at 8°C for up to 2 weeks. When cultures kept at 8°C for up to 30 days were transferred to 28°C, growth resumed quickly, and at a rate virtually identical to that for unchilled cells. Similar results were found for cells maintained at 0°C, and for cells of `VFNT Cherry' and of L. hirsutum. Under certain conditions, cultures slowly doubled in fresh weight and cell volume at 8 or 9°C but additional growth at 8°C did not occur, nor could growth be maintained by subculture at 8 or 9°C. The results are contrary to reports that cell cultures of tomato die when exposed to temperatures below 10°C for 1 or 2 weeks. Our observations indicate that chilling temperatures quickly inhibit growth of tomato cells, but do not kill them.     

REVERSIBLE, THERMOTROPIC ALTERATION OF NUCLEAR MEMBRANE STRUCTURE AND NUCLEOCYTOPLASMIC RNA TRANSPORT IN TETRAHYMENA

We examine the effect of cooling upon the freeze-etch ultrastructure of nuclear membranes, as well as upon nucleocytoplasmic RNA transport in the unicellular eukaryote Tetrahymena pyriformis. Chilling produces smooth, particle-free areas on both faces of the two freeze-fractured macronuclear membranes. Upon return to optimum growth temperature the membrane-associated particles revert to their normal uniform distribution and the smooth areas disappear. Chilling lowers the incorporation of [¹⁴C]uridine into whole cells and their cytoplasmic RNA. Cooling from the optimum growth temperature of 28° to 18°C (or above) decreases [¹⁴C]uridine incorporation into cells more than into their cytoplasmic RNA; chilling to below 18°C but above 10°C causes the reverse. [¹⁴C]Uridine incorporation into whole cells and their cytoplasmic RNA reflects overall RNA synthesis and nucleocytoplasmic RNA transport, respectively. RNA transport decreases strongly between 20° and 16°C, which is also the temperature range where morphologically detectable nuclear membrane transitions occur. This suggests that the nuclear envelope limits the rate of nucleocytoplasmic RNA transport at low temperatures. We hypothesize that a thermotropic lipid phase transition switches nuclear pore complexes from an "open" to a "closed" state with respect to nucleocytoplasmic RNA transport.     

Photosynthetic and respiratory rates of two psychrophilic diatoms

The photosynthetic rates in two psychrophilic diatoms, Chaetoceros sp. strain K3-10 and Nitzschia sp. K3-3 for cells grown at 0°C were 8 to 10 microliters O₂ evolved per milligram dry weight per hour, and 10-fold higher, about 80 for cells grown at 10°C. The respiration rates followed the same pattern, with a value of around 1 microliter dark uptake per milligram dry weight per hour for both organisms grown at 0°C, and 6 to 10 for cells grown at 10°C. When cells grown at 0°C were immediately shifted to 10°C or cells grown at 10°C were shifted to 0°C, the respiratory rates quickly adapted to values characteristic of cells grown at the shift temperature. On the other hand, the light-saturated rate of O₂ evolution showed much less immediate adaptation, especially on the up shift, 0° to 10°C. The chlorophyll a content of 0°C grown cells was about 0.5% of dry weight, in 10°C grown cells 1.3% (strain K3-10) and 2.2% (strain K3-3). In addition to a diminished chlorophyll a content in 0°C grown cells, there seemed proportionally (by absorbance and calculation) less c to a than in 10°C grown cells. The relative fluorescence excitation spectra of 680-nm emission also showed a lower contribution by both chlorophyll c and fucoxanthin in 0°C grown cells of Chaetoceros sp. strain K3-10 as compared to 10°C grown cells. The data at hand suggest that in psychrophilic diatoms continuously growing at 0°C there may be problems associated with synthesis of an effective accessory pigment system, and as a working hypothesis it is suggested this is related to restriction of synthesis of one or several accessory pigment proteins.     

Storage Temperature Alters the Expression of Differentiation-Related Genes in Cultured Oral Keratinocytes

Purpose

Storage of cultured human oral keratinocytes (HOK) allows for transportation of cultured transplants to eye clinics worldwide. In a previous study, one-week storage of cultured HOK was found to be superior with regard to viability and morphology at 12°C compared to 4°C and 37°C. To understand more of how storage temperature affects cell phenotype, gene expression of HOK before and after storage at 4°C, 12°C, and 37°C was assessed.

Materials and Methods

Cultured HOK were stored in HEPES- and sodium bicarbonate-buffered Minimum Essential Medium at 4°C, 12°C, and 37°C for one week. Total RNA was isolated and the gene expression profile was determined using DNA microarrays and analyzed with Partek Genomics Suite software and Ingenuity Pathway Analysis. Differentially expressed genes (fold change > 1.5 and P < 0.05) were identified by one-way ANOVA. Key genes were validated using qPCR.

Results

Gene expression of cultures stored at 4°C and 12°C clustered close to the unstored control cultures. Cultures stored at 37°C displayed substantial change in gene expression compared to the other groups. In comparison with 12°C, 2,981 genes were differentially expressed at 37°C. In contrast, only 67 genes were differentially expressed between the unstored control and the cells stored at 12°C. The 12°C and 37°C culture groups differed most significantly with regard to the expression of differentiation markers. The Hedgehog signaling pathway was significantly downregulated at 37°C compared to 12°C.

Conclusion

HOK cultures stored at 37°C showed considerably larger changes in gene expression compared to unstored cells than cultured HOK stored at 4°C and 12°C. The changes observed at 37°C consisted of differentiation of the cells towards a squamous epithelium-specific phenotype. Storing cultured ocular surface transplants at 37°C is therefore not recommended. This is particularly interesting as 37°C is the standard incubation temperature used for cell culture.