Assessment of hair follicle viability in artificial media before transplantation

Follicular viability and histological assessment after cryopreservation of whole sheep ovaries with vascular pedicle by vitrification

Fertility preservation strategies warrant non-invasive viability assessment of preantral follicles (PAF) such as staining with Neutral Red (NR) that is incorporated by viable follicles. To optimize the procedure, we firstly determined the lowest concentration and shortest exposure time needed for optimal viability screening of isolated bovine PAF. Secondly, we combined this protocol to a vitrification procedure to assess cryotolerance of the stained follicles. Isolated PAF (900, divided over 6 replicates) were cultured in DMEM/Ham's F12 (Culture Medium - Cm) for 4 days (38.5 °C, 5% CO2). On D0, D2 and D4, follicles were stained, by adding NR medium (NRm = Cm with different concentrations NR) after which viability was assessed by counting stained/non-stained PAF every 30 min for a period of 2 h. Following a binary logistic regression analysis with staining as a result (yes/no) versus log-concentration, a probability model could be fitted, indicating that the proportion of stained follicles remained stable after 30 min when 15 μg/ml NR was used, without compromising follicular health and viability. Consequently, using this protocol, no significant effect of staining prior to vitrification, was found on PAF viability immediately after warming or following 4 days of culture. In conclusion, we propose NR staining as a non-invasive, non-detrimental viability assessment tool for PAF, when applied at 15 μg/ml for 30 min, being perfectly compatible with PAF vitrification.

Morphologic, ultrastructural, and biochemical identification of apoptosis in vitrified-warmed mouse ovarian tissue

The incidence of young women diagnosed with cancer has been globally increasing. In many cases the surgical approach followed by chemotherapy, radiotherapy or hormonal therapy could lead to infertility or premature ovarian failure. Several options are available in order to preserve fertility and increase the future gestation rate. Among embryo cryopreservation and oocyte cryopreservation, ovarian tissue cryopreservation represents an ideal option, especially for premenopausal women and for those who cannot delay the start of chemotherapy. The purpose of this study was to examine the follicle viability using fluorescence microscope before and after ovarian thawing.

P-474: Assessment of follicular viability, apoptotic markers and blood vessels status after cryopreservation of intact human ovary with its vascular pedicle

Recent improvement in neuroscience has led to new strategies in neural repair. Hair follicle stem cells are high promising source of accessible, active, and pluripotent adult stem cells. They have high affinity to differentiate to neurons. Aside from using cell-scaffold combinations for implantation, scaffolds can provide a suitable microenvironment for cell proliferation, migration, and differentiation. NT-3 is the most interesting neurotrophic factors being an important regulator of neural survival and differentiation. Since treatment duration in neural repair is very important, this study aims to evaluate the effect of NT-3 and poly-L-lactic acid (PLLA) on differentiation time of bulge stem cells of rat hair follicle to neural-like cells. HFSCs of rat whisker was isolated and cultured on PLLA and differentiated with 10ng/mL NT-3. Biological features of cultured cells were evaluated with immunocytochemistry and flowcytometry methods by using CD34, nestin, and βІІІ-tubulin markers. For cell viability and morphological assessment, MTT assay and SEM were performed. Our results showed that bulge stem cells of hair follicle can express CD34 and Nestin before differentiation. By using NT-3 during differentiation process, the cells showed positive reaction to βІІІ-tubulin antibody. MTT results demonstrated that PLLA significantly increased cell viability. Finally, HFSCs adhesion was confirmed by SEM results. The results indicate that 10ng/mL NT-3 and PLLA have significant effect on differentiation time of rat HFSCs to neural cells even in 10days.

Recent improvement in neuroscience has led to new strategies in neural repair. Hair follicle stem cells are high promising source of accessible, active, and pluripotent adult stem cells. They have high affinity to differentiate to neurons. Aside from using cell-scaffold combinations for implantation, scaffolds can provide a suitable microenvironment for cell proliferation, migration, and differentiation. NT-3 is the most interesting neurotrophic factors being an important regulator of neural survival and differentiation. Since treatment duration in neural repair is very important, this study aims to evaluate the effect of NT-3 and poly-L-lactic acid (PLLA) on differentiation time of bulge stem cells of rat hair follicle to neural-like cells. HFSCs of rat whisker was isolated and cultured on PLLA and differentiated with 10 ng/mL NT-3. Biological features of cultured cells were evaluated with immunocytochemistry and flowcytometry methods by using CD34, nestin, and βІІІ-tubulin markers. For cell viability and morphological assessment, MTT assay and SEM were performed. Our results showed that bulge stem cells of hair follicle can express CD34 and Nestin before differentiation. By using NT-3 during differentiation process, the cells showed positive reaction to βІІІ-tubulin antibody. MTT results demonstrated that PLLA significantly increased cell viability. Finally, HFSCs adhesion was confirmed by SEM results. The results indicate that 10 ng/mL NT-3 and PLLA have significant effect on differentiation time of rat HFSCs to neural cells even in 10 days.

A comparison of isolation techniques for small preantral follicles (30-70 microm) from bovine ovaries using a mechanical method with a grating device or collagenase treatment was performed. The mean number (157.0) of intact follicles per ovary isolated by the mechanical method was significantly greater (P < 0.05) than that (26.0) of follicles isolated by the enzymatic method. Isolated morphologically normal follicles (MNF) were cultured for up to 30 d either in control cultures (non-coculture) or in coculture with bovine ovary mesenchymal cells (BOM), fetal bovine skin fibroblasts (FBF), and/or bovine granulosa cells (BGC). In control cultures, most of the follicles degenerated and only a few MNF (1.2%) were present after 30 d in culture. In contrast, the cocultures with BOM, FBF, and BGC resulted in 50.7, 46.6, and 21.4% viable MNF, respectively. Trypan blue and Hoechst 33258 staining were used for a quick and sensitive assessment of oocyte and granulosa cell viability during follicle isolation and culture in vitro. After 30 d, percentages of viable follicles in coculture with BOM (18.6%) and FBF (17.1%) were significantly greater than those of follicles in the control cultures (0%) or in coculture with BGC (10.0%). There was a gradual increase in the average diameter of the MNF during culture. The mean diameter of the follicles increased by 15.4 and 30.0% in coculture with BOM and FBF, respectively, by day 30. In conclusion, small bovine preantral follicles were efficiently isolated using a mechanical method that utilizes a grating device, and could be maintained for up to 30 d in the presence of mesenchymal cell cocultures such as BOM and FBF. This in vitro culture system that supports long-term survival of bovine preantral follicles should be beneficial for studying follicle growth and development.

Role of media, protein and energy supplements on maintenance of morphology and DNA-synthesis of small preantral domestic cat follicles during short-term culture

Background Several surgical treatment methods have been proposed for the treatment of stable vitiligo, but they remain inadequate in terms of patient satisfaction. Objective To assess the efficacy and safety of a modified autologous cultured hair follicle outer root sheath (HF-ORS) cell suspension transplantation in the treatment of stable vitiligo lesions, and to compare it with transplantation of autologous non-cultured HF-ORS cell suspension for the same patient. Patients and methods Twenty-four patients were enrolled in this prospective controlled study. Hairs were epilated, and HF-ORS cell suspensions were prepared using two methods for the same patient. The recipient site was prepared by CO2 laser resurfacing. Assessment of cellular activity and viability included cellular melanin content determined by enzyme-linked immunosorbent assay and measurement of the fold change in premelanosome (Pmel-17) gene expression. The patients were followed-up for 6 months postprocedure. Results Nineteen patients completed the study. The results demonstrated that the cultured method showed superior repigmentation response in 15 (79%) patients, three (16%) responded equally, and one (5%) was more tolerant of the noncultured method. Conclusion Cultured autologous HF-ORS melanocytes show superior repigmentation response, increased melanin content, and upregulated gene expression of Pmel-17 than that of noncultured ones. However, the study also demonstrated that both methods are potential options for the treatment of stable vitiligo.

Cultivation of Murine Hair Follicles as Organoids in a Collagen Matrix

Freeze-thawing intact human ovary with its vascular pedicle with a passive cooling device

ABSTRACTAim:The purpose of this in vitro hair graft study was to better understand how different storage media affect the survival of hair follicles at different temperatures during a hair transplant procedure.Materials and Methods:In this study, hair follicles (n = 60) were harvested from 3 healthy male volunteers. Follicles were randomly assigned to the following groups: Group A: Storage media phosphate-buffered saline (PBS), platelet-rich plasma (PRP), platelet-rich fibrin (I-PRF), and Dulbecco’s Modified Eagle Medium (DMEM). Group B: Storage media were placed at the following temperatures: 4°C, 26°C (room temperature), and 37°C (body temperature). The viability of hair follicles was checked using the explant culture method. Cell outgrowth was observed after incubation at 37°C in DMEM containing 10% FBS.Results:Explant cultures of hair follicles stored at 4°C and 26°C did not show outgrowth of the cells after 7 days of culture. Explant cultures of hair follicles stored at 37°C did show outgrowth of the cells after 7 days of culture. Furthermore, these study results indicated that 10% DMEM preserves hair follicles more effectively than PBS, PRP, and I-PRF.Conclusion:According to the study’s findings, freezing graft storage options might not be the best option. Instead of 4°C or room temperature 26°C, 37°C has been shown to improve hair graft survival. Furthermore, these study results indicated that 10% DMEM preserves hair follicles more effectively than PBS, PRP, and I-PRF. The study concluded that maintaining hair follicles in 10% DMEM at 37°C would prolong graft life and improve therapeutic outcomes.

Hair follicle preservation for the purpose of delayed application would help us to transplant hair follicles more efficiently. Isolated single hair follicles were preserved at 4 degrees C in four different solutions. Viability of preserved follicles was judged by organ culture and cell culture. In addition, a small number of hair follicles were transplanted into athymic mice. RESULTS. By cell culture, both dermal papilla and outer root sheath cells could be cultivated after 7 days of preservation. Hair follicles preserved for 48 hours showed a significant increase of hair shafts in organ culture. Those preserved for 7 days regrew well when transplanted into athymic mice. Preservation of hair follicles at 4 degrees C could be one option to prepare many follicular units at one time for transplantation.

Biotin deficiency is caused by inflammatory bowel diseases that impair the absorption of the vitamin, special dietary disorders with the consumption of raw eggs (excess avidin – a vitamin B7 blocker protein), magnesium deficiency, smoking, alcohol, treatment with broad-spectrum antibiotics, sulfonamides, and anticonvulsants. Hypovitaminosis B7 has also been noted in individuals with congenital genetic defects of the biotinidase gene or other genes involved in biotin metabolism. Deficiency of water-soluble vitamin B7 (vitamin H) – manifested by dry skin, seborrheic dermatitis, dermatitis around the eyes, nose, mouth, ears and groin, impaired nail growth, slow healing of skin cuts, atopic dermatitis, striations, splitting, brittle nails and alopecia (diffuse and androgenic form). Alopecia occurs when hair follicles die and leads to hair loss. The human proteome contains 51 proteins involved in biotin metabolism. In particular, D-biotin-dependent carboxylases play an important role in the metabolism of fatty acids, amino acids, carbohydrates, cell division and growth, incl. keratinocytes and hair follicle cells. The molecular mechanisms of the effects of D-biotin on the skin and its appendages may involve various growth factors: regulation of the signaling pathways of growth factors (IGF-1, FGF, KGF, HGF, VEGF, SIRT-1, Wnt and beta-catenin) has been shown. Hair follicle stem cells cause the cyclical growth of hair follicles. Growth factors are involved in the activation of stem cell growth by D-biotin; activation of the Wnt/β-catenin signaling cascade leads to the activation of cyclin D1 proteins (initiates DNA synthesis and leads to increased viability of hair follicles. The results of fundamental and clinical studies confirm the prospects of using biotin in dermatology for the treatment of diseases of the skin, hair and nails, incl. alopecia of various origins (androgenic, focal, diffuse). The results of the studies indicated that biotin was well tolerated, and there was no risk of hypervitaminosis even when taking megadoses (hundreds of milligrams).