Human serum albumin as a clinically accepted cell carrier solution for skin regenerative application

The rules governing Medicinal Products in the European Union necessitates the production of cell-based therapy in good manufacturing practice facilities. The produced cells may need several hours in transportation to reach the application sites. In this study, we investigated four candidate solutions for transporting human keratinocytes. The solutions are (1) normal saline, (2) saline with 2.5% human serum albumin (Saline + HSA), (3) chemically defined, xeno-free keratinocyte media and (4) keratinocyte media with pituitary bovine extract (PBE-media). One million keratinocytes from three donors were suspended in each solution and kept at 4 °C for up to 24 h. Cells kept in Saline + HSA showed higher viability after 1, 3 and 24 h. Then, equal number of viable cells were seeded on collagenous matrix and cultured for 48 h. The adhesion and colonization were higher in the cells kept in PBE-media, while the keratinocyte surface marker, cytokeratin 14, was present in all studied groups. These results confirmed the suitability of Saline + HSA as a cell transportation solution for clinical use, which will be the choice for the planned clinical trial. Keratinocyte PBE-media can be an alternative for cells transported for research purpose, if the same media type is going to be used in the following experiments.

www.nature.com/scientificreports/ application sites. Furthermore, the readiness of the patient and the surgical team for application might require extra time, which can be extended up to 24 h. It is preferred to transport the cell product as fresh cells since freezing the cells can be associated with decreased cell numbers and phenotype. Furthermore, quality control could be required after thawing, which could affect the certified release criteria and cannot be conducted outside the manufacturer site [5][6][7] . The debate about the optimum cell carrier/transport solution was focused on maintaining the viability of the cells without affecting their phenotype or functionality, for this time span. This study evaluated four candidate solutions for transporting keratinocyte at 4 °C for 24 h, which included (1) normal saline solution (0.9% NaCl); (2) saline with 2.5% human serum albumin (Saline + HSA); (3) chemically defined, xenofree keratinocyte media (CDM), which will be used for keratinocyte expansion for clinical use; and (4) keratinocyte media with bovine pituitary extract (BPE-media), the classically used media for keratinocyte expansion in research.

Results
Keratinocytes viability following preservation at + 4 °C. Keratinocytes were kept in the corresponding transport solution at + 4 °C for 24 h. The cell count and viability were determined after 1 h, 3 h and 24 h (Fig. 1). After 1 hour, cells suspended in saline + HSA showed higher total cell number (163% of the saline group) than the rest of the groups, while the number of viable cells was higher (244%) than among those suspended in saline (100%) and CDM (81%). Similarly, cells kept in Saline + HSA showed higher viability (152%) than CDM (95%) with a similar tendency with BPE-media (101%). After 3 h, the number of viable cells was higher in Saline + HSA (295%) and BPE-media (182%) groups than the saline (100%) and CDM (89%) groups. The cell viability was lower in CDM than the rest of the groups. BPE-media had higher viability than the saline group and the cells suspended in Saline + HSA showed a similar tendency.
After 24 h, the number of viable cells (225%) as well as the cell viability (142%) were higher in the Saline + HSA group in comparison to the saline (100%; 100%) and CDM (83%; 96%) groups and with a similar tendency in comparison with BPE-media (139%; 107%).
Total cell number was higher in the Saline + HSA group in comparison to the saline and CDM groups at all studied timepoints, as well as BPE-media group after 3 h with a similar tendency after 1 h. The latter group had significant higher total cell number than saline after 1 h and 24 h and a similar tendency at 3 h. No difference was detected between the total cell number of the saline and CDM groups at any of the studied timepoints. The cell counting and viability was related to that of the saline group in each sample. The total cell number (A) was higher in the saline + HSA group after 1 and 24 h in comparison to saline and CDM groups and higher than all studied groups at 3 h. BPE-media was higher in comparison to saline after 1 and 24 h and more than CDM at 24 h. The viable cell number (B) was similarly higher in saline + HSA in comparison to saline and CDM at all studied timepoints, with a similar trend with BPE-media. The cell viability (C) was higher in saline + HSA group in comparison to CDM at all timepoints and to saline at 24 h. BPE-media had higher viability than saline and CDM groups at 3 h. n = 3. www.nature.com/scientificreports/ Keratinocyte adhesion and colonization after preservation. After 24 h in the corresponding transport solution, the cells were resuspended and equal number of viable cells were cultured at 37 °C and 5% CO 2 , for 48 h. The cells were then fixed and stained with crystal violet (Fig. 2). The viable cells from all transport solutions kept their ability to adhere to the culture surface, as well as colony formation. While visual difference of the staining between the groups was not clear, elution of the stain showed higher absorbance for the cells in PBE-media (191%) in comparison to all other groups, as well as between CDM (135%) and saline (100%). The was no difference between CDM and saline + HSA (117%).

Keratinocytes expression of the surface marker cytokeratin 14 and involucrin. Keratinocytes
were fixed after 48 h culture and characterized by immuno-staining of the keratinocyte markers cytokeratin 14 (CK-14) or involucrin. The cells from all suspension groups were positive for the stain, and no difference could be observed between the groups (Fig. 3).

Discussion
Cell based therapy is an upcoming field in modern medical management of many conditions, including skin wounds 8,9 . The European Directorate for the Quality of Medicines & Health Care has considered cultured cells as an advanced medicinal product, which needs extensive validation. The whole manufacturing process needs to comply to the regulations of the European Medicines Agency (EMA), including the transportation procedure. While the temperature and sterility chain are well described, the transport solution has yet to be established. This solution should provide a vehicle that maintain the cell phenotype, function and viability over the transportation time from the manufacturing to the application sites. Furthermore, the cells may sometimes have to be kept overnight when the application is not immediately possible. The transportation of cells is not only limited  www.nature.com/scientificreports/ to ATMP or research but also for diagnostic purposes. In an interesting study, the authors shipped whole blood samples overnight, in their collection tubes, to a distant laboratory and investigated the possible effect on the immune cells. While T lymphocytes showed mild shift of their subclasses, B lymphocytes and natural killer cells had altered production of certain cytokines 10 . In Linköping University Hospital, a plan is undergoing to treat burn patients and chronic wounds patients with autologous sprayed keratinocytes. The cells will be manufactured by the Vecura GMP facility in Stockholm and will need to be transferred to the premises in Linköping, about 200 km south-east of Stockholm. Ideally, the cells will be applied immediately but, according to the patient condition and the availability of the surgical team, a delay for up to 24 h could be expected. A multidisciplinary team had discussions regarding the best vehicle for transporting the cells and preserving them overnight. The suggestions included having a balanced solution, such as normal saline, or cell culture media. The later should have the advantage of providing necessary nutrients to the cells. Some laboratories transfer or exchange cells just by shipping living cells in fresh cell culture media, in sterile container and in ambient temperature. In this study site, two types of media are used for culturing keratinocytes; the first is described as serum free media which has bovine pituitary extract among other components. This media is not recommended to be used for manufacturing cells for clinical application due to the risk of transferring diseases between animals and humans. Instead, a xeno-free media is being used for such purpose. Saline is the most used balanced solution, that can be readily accessible as a authorized medicinal product. Lymphoid tissue was freshly transported in saline at refrigerator temperature and the immunophenotyping has been stable for 7 days 11 . In order to provide the saline with a protective vehicle, 2.5% of HSA was added to the solution. HSA was used as a substitute for fetal calf serum in cell microencapsulation in poly lysine alginate for biomedical application 12 . This composition is common for drug delivery solutions for injection 13 . In addition it has been used as a preservative solution, at the same concentration, for endothelial and smooth muscle cells 14 .
In the latter study the authors investigated different concentrations of human serum albumin, in comparison to normal saline, to preserve the cells for 24 and 48 h at 4 °C. The authors concluded that 2.5% is the optimum concentration, as proved by the viability of the cells and their adherence after preservation. In another study, Lee et al. found that adding 10% HSA to saline or Dulbecco's Modified Eagle Medium would enhance the viability of gingival-derived oral keratinocytes when kept at 4 °C. The cells kept the same distribution among different phases of the cell cycle up to 48 h, but the authors did not show any functional or phenotypic effects. Interestingly, there was no difference in the cell viability when the cells were kept in saline with 10% HSA either in 4 °C or 25 °C 15 .
The production of recombinant HSA overcame the availability and potential infectivity problems that can be associated with this important protein. HSA is widely used as a balanced drug carrier in solutions, as well as in more complicated drug delivery systems 16,17 . Albumin is incorporated into the cell culture in conjunction with growth factors and other peptides. Additionally, this small protein can bind to the external surface of the cell membrane, through non-specific adsorption, and provides cell protection. This role is particularly evident in bioreactors, where albumin prevent the physical damage of the cells caused by hydrodynamic stress. Furthermore, albumin can be internalized to the cells by endocytosis, through clathrin-coated pits 12 . To the best of our knowledge, this effect has not been studied in keratinocytes.
Cell culture media was used as a transport solution for avulsed teeth, which can keep the viability of the tooth for 48 h. After reimplantation, the periodontal ligament cells were viable during 12 months of follow up 18 . Similarly, fibroblast sheets were transported in cell culture media for 8 h without thermal regulation (20-30 °C), on ice (5-10 °C) and in a special container that keeps the temperature at 37 °C, without significant difference in the histological morphology of the layers, in comparison to the control. The latter was kept in the standard CO 2 incubator 19 . In a similar experiment, mucosal epithelial sheets were transported for 12 h in four different transportation media; keratinocyte culture media, keratinocyte culture media without fetal calf serum, DMEM/ F12 media with epidermal growth factor and Hanks balanced salt solution. The viability of the cells upon delivery was 89.9%, 93.8%, 87.1% and 90.7%, which indicated no significant difference between the studied solutions. The morphology of the cell sheets was similar before and after transportation 20 . These reports confirmed the validity of the cell culture media in the transportation of living chilled cells, which matched our findings. Cells kept at 4 °C in cell culture media were still viable and able to adhere to cell culture plastic.
On the other hand, BPE was added as an essential component of keratinocytes culture media as BPE enhanced proliferation and helped to maintain their phenotype 21 . The protein component of BPE is cell-protective against oxidative stress. Its mitogenic ability was estimated to be 70 times higher than serum because of the presence of a cocktail of growth factor, such as growth hormone, basic fibroblast growth factor, epidermal growth factor like proteins, and platelet-derived growth factor 22 . In our results, the higher colonization ability with PBE-media group could reflect the effect of these factors. In this case, the safety of the PBE-media as a carrier solution would be questioned. Despite the excessive wash, the cells had higher ability to adhere and proliferate, which could reflect either binding of the bovine growth factors to the human cells or-at least-stimulating the corresponding signaling pathway(s). In the first case, there will be a risk of transmitting an animal protein to the patient. Hypersensitivity can be a consequence with failure of cell transplantation. Such effect is well known for patients received cultured cells in the presence of fetal calf serum 23,24 . Besides, the transmission of infectious agents from the animal derived raw materials to humans is a health risk, especially with prions 12 . In the second case of complete removal of animal-derived protein, the uncontrolled mitogenic stimulation of these pathways would need detailed investigations for their safety, on the short and long terms.
In conclusion, this study recommends the use of 2.5% HSA in saline as a transportation vehicle for keratinocytes for up to 24 h at 4 °C for clinical use. The research team decided, after several discussions, to use this solution for the cell transportation of the proposed clinical trial based on the perseverance of cell viability as well as the phenotype. Furthermore, the components of this solution can be obtained as authorized medicinal products that is compliant to EMA guidelines. The ultimate evaluation of our findings will be reflected by the success rate of keratinocyte application in clinical studies. In the meantime, keratinocyte media with PBE can crystal violet assay. Fixed cells were rinsed with phosphate buffered saline (PBS) and incubated for 10 min at room temperature with 0.5% crystal violet solution (Sigma-Aldrich, USA). The cells were excessively washed with running water. Cell adherence and colonization was examined using an inverted microscope and imaged with an AxioCam ERc5s camera (Carl Zeiss Microscopy, Germany). Then the stain was eluted in 10% acetic acid (Sigma-Aldrich, USA) and intensity was evaluated spectrophotmetricaly at 595 nm (Spectramax plus 384, Molecular Devices, USA).
immunocytochemistry. The fixed keratinocytes underwent antigen retrieval using preheated tris-urea buffer (Thermo Scientific, USA) at 95 °C. The cells were permeabilized by incubation for 15 min at room temperature with 0.05% Triton X-100 (Thermo Scientific, USA). Following blockage, anti-cytokeratin 14 or anti-involucrin (Abcam, UK) at 1 μg/ml was incubated overnight on a rocking stage at 4 °C. Then the cells were washed, and the mouse and rabbit specific secondary antibody with horseradish peroxidase/3-amino-9-ethylcarbazole detection immunohistochemistry kit (Abcam, UK) was used according to the manufacturer's instruction and counterstained with Alcian blue solution (Sigma-Aldrich, USA). The stained cells were imaged using inverted microscopy, as described above.