A novel system for expansion and delivery of human keratinocytes for the treatment of severe cutaneous injuries using microcarriers and compressed collagen.

Cell therapy with autologous or allogeneic keratinocytes applied as a single-cell suspension is well established in clinical practice in the treatment of severe burn injuries to augment epithelial barrier restoration. Yet, the application of cell sprays can lead to significant cell loss owing to lack of adhesion of cell suspension to the wound bed. The development of a robust and controllable method of transplanting cells onto the wound bed is yet to be established. The ability to control adhesion and distribution of cells by using a cell carrier embedded in a biodegradable scaffold could significantly improve the treatment of cutaneous wounds with keratinocyte cell therapy. Several microcarrier-based systems for expanding keratinocytes already exist. A new method for expansion of human keratinocytes in a feeder-free, defined medium system on microcarriers has been developed. The cells retained their basal, proliferative phenotype after rapid expansion in a clinically relevant time-frame. The cell-laden microcarriers were further incorporated into collagen scaffolds fabricated by plastic compression. When cultured in vitro, cells continued to proliferate and migrate along the surface of the collagen scaffold. Using an in vitro wound bed model, cells were observed to form mostly single cell layers and in some areas multiple cell layers within 8 days, while retaining their basal, proliferative phenotype, indicating the suitability of this cell transplantation method to improve epithelial barrier restoration. This advanced cell expansion and delivery method for cutaneous cell therapy provides a flexible tool for use in clinical application. Copyright © 2017 John Wiley & Sons, Ltd.

Immune reconstitution inflammatory syndrome in CNS tuberculosis.

BACKGROUND: Immune Reconstitution Inflammatory Syndrome (IRIS), an exaggerated inflammatory response with clinical worsening due to immune recovery during treatment, is rare in the immune-competent population. CASE CHARACTERISTICS: A 5-½-year old immune-competent girl with CNS tuberculosis without HIV who developed paradoxical IRIS. OUTCOME: Response to supportive care along with Anti-tuberculosis treatment. MESSAGE: IRIS can occur in tuberculosis, even in the immuno-competent.

A fully autologous co-culture system utilising non-irradiated autologous fibroblasts to support the expansion of human keratinocytes for clinical use.

Autologous keratinocytes can be used to augment cutaneous repair, such as in the treatment of severe burns and recalcitrant ulcers. Such cells can be delivered to the wound bed either as a confluent sheet of cells or in single-cell suspension. The standard method for expanding primary human keratinocytes in culture uses lethally irradiated mouse 3T3 fibroblasts as feeder cells to support keratinocyte attachment and growth. In an effort to eliminate xenobiotic cells from clinical culture protocols where keratinocytes are applied to patients, we investigated whether human autologous primary fibroblasts could be used to expand keratinocytes in culture. At a defined ratio of a 6:1 excess of keratinocytes to fibroblasts, this co-culture method displayed a population doubling rate comparable to culture with lethally irradiated 3T3 cells. Furthermore, morphological and molecular analysis showed that human keratinocytes expanded in co-culture with autologous human fibroblasts were positive for proliferation markers and negative for differentiation markers. Keratinocytes expanded by this method thus retain their proliferative phenotype, an important feature in enhancing rapid wound closure. We suggest that this novel co-culture method is therefore suitable for clinical use as it dispenses with the need for lethally irradiated 3T3 cells in the rapid expansion of autologous human keratinocytes.

The effect of pH in modulating skin cell behaviour.

BACKGROUND: Variations occur in the pH of cutaneous wounds which may affect wound closure, graft take and microbial infection rates. OBJECTIVES: To determine how pH modulates cell behaviour in order to optimize wound care. METHODS: The effects of pH on the attachment, proliferation and migration of keratinocytes and fibroblasts were investigated in vitro and in an ex vivo skin growth model. In addition, the effect of pH on keratinocyte differentiation as measured by the expression of cytokeratins 1 (K1) and K5 was studied. RESULTS: We demonstrated that the optimal pH for both keratinocyte and fibroblast proliferation is between pH 7.2 and 8.3. The optimal pH for growth from ex vivo skin explants was pH 8.43 which correlates with a previously reported improvement in skin graft take at higher pHs. Expression of K1 was found to be elevated in keratinocytes at a low pH. CONCLUSIONS: These results demonstrate that skin cells and explants proliferate and migrate at pHs higher than the physiological pH and that at lower pH keratinocytes express a differentiated keratinocyte phenotype. A better understanding of the responses of the cellular components of skin to fundamental physiological variables such as pH may help inform improved clinical wound care.