Feeder layer- and animal product-free culture of neonatal foreskin keratinocytes: improved performance, usability, quality and safety

Since 1987, keratinocytes have been cultured at the Queen Astrid Military Hospital. These keratinocytes have been used routinely as auto and allografts on more than 1,000 patients, primarily to accelerate the healing of burns and chronic wounds. Initially the method of Rheinwald and Green was used to prepare cultured epithelial autografts, starting from skin samples from burn patients and using animal-derived feeder layers and media containing animal-derived products. More recently we systematically optimised our production system to accommodate scientific advances and legal changes. An important step was the removal of the mouse fibroblast feeder layer from the cell culture system. Thereafter we introduced neonatal foreskin keratinocytes (NFK) as source of cultured epithelial allografts, which significantly increased the consistency and the reliability of our cell production. NFK master and working cell banks were established, which were extensively screened and characterised. An ISO 9001 certified Quality Management System (QMS) governs all aspects of testing, validation and traceability. Finally, as far as possible, animal components were systematically removed from the cell culture environment. Today, quality controlled allograft production batches are routine and, due to efficient cryopreservation, stocks are created for off-the-shelf use. These optimisations have significantly increased the performance, usability, quality and safety of our allografts. This paper describes, in detail, our current cryopreserved allograft production process

Glycerol treatment as recovery procedure for cryopreserved human skin allografts positive for bacteria and fungi

Human donor skin allografts are suitable and much used temporary biological (burn) wound dressings. They prepare the excised wound bed for final autografting and form an excellent substrate for revascularisation and for the formation of granulation tissue. Two preservation methods, glycerol preservation and cryopreservation, are commonly used by tissue banks for the long-term storage of skin grafts. The burn surgeons of the Queen Astrid Military Hospital preferentially use partly viable cryopreserved skin allografts. After mandatory 14-day bacterial and mycological culture, however, approximately 15% of the cryopreserved skin allografts cannot be released from quarantine because of positive culture. To maximize the use of our scarce and precious donor skin, we developed a glycerolisation-based recovery method for these culture positive cryopreserved allografts. The inactivation and preservation method, described in this paper, allowed for an efficient inactivation of the colonising bacteria and fungi, with the exception of spore-formers, and did not influence the structural and functional aspects of the skin allografts

HIV transmission by transplantation of allograft skin: a review of the literature.

The fear of human immunodeficiency virus (HIV) transmission by means of allograft skin has led to a cautious approach to allograft donor selection. However, no irrefutable diagnostic test exists to determine the possible presence of HIV at the time of donation. In order to find ways of improving HIV donor screening practices for skin banks, we review the presence of HIV in human skin, explore the possible transmission of HIV by transplantation of human allograft skin, and discuss the reliability of existing HIV tests. The use of the polymerase chain reaction (PCR) as a sensitive detection system for HIV infection of skin biopsies, in combination with conventional routine HIV blood screening tests; could lower the risk of transmitting HIV to severely burned patients

Lyophilized keratinocyte cell lysates contain multiple mitogenic activities and stimulate closure of meshed skin autograft-covered burn wounds with efficiency similar to that of fresh allogeneic keratinocyte cultures.

For several years, grafting with allogeneic keratinocyte cultures has been used successfully as a wound-healing therapy both by us and by many other groups. Since their postgrafting survival time is limited, the effect of these cultures is generally explained by the production of wound repair-stimulating factors that promote proliferation and migration of resident cells. In this study we show that lysates of cultured keratinocytes contain mitogenic activity for keratinocytes, endothelial cells, and fibroblasts. In addition, the lysates inhibit the contraction of collagen gels by human skin fibroblasts. On the basis of these observations and of in vivo data obtained by ourselves and others, we have evaluated the effect of total keratinocyte lysates on the healing of meshed skin autograft-covered burn wounds. Twenty burn wounds were tangentially excised and autografted with one to three meshed conventional skin transplants. An area treated with a gel containing lysated keratinocyte cultures was compared with an area treated with placebo-gel in terms of epithelialization on day 5. In six patients an additional fresh keratinocyte alloculture was applied as a positive control. Results indicate that the newly formed epithelium (difference between percentage of epithelialization on day 5 and on day 0) was 31.1 percent in the treated area compared with 16.5 percent in the placebo area. This result is comparable with the value obtained by treatment with fresh keratinocyte allocultures, namely, 33.8 percent. These figures show a twofold stimulation of epithelialization

High frequency percussive ventilation and conventional ventilation after smoke inhalation: a randomised study.

Inhalation injury and bacterial pneumonia represent some of the most important causes of mortality in burn patients. Thirty-five severely burned patients were randomised on admission for conventional ventilation (CV; control group) versus high frequency percussive ventilation (HFPV; study group). HFPV is a ventilatory mode, introduced 10 years ago which combines the advantages of CV with some of those of high frequency ventilation. Arterial blood gases, ventilatory and hemodynamic variables were recorded for 5 days at 2h intervals. Incident complications were classically managed. A statistical analysis (Student's t-test and Wilcoxon signed rank test) demonstrated a significant higher PaO(2)/FiO(2) from days 0 to 3 in the HFPV group. No significant differences were observed for the other parameters. Our findings suggest that HFPV can improve blood oxygenation during the acute phase following inhalation injury allowing reduction of FiO(2). No significant differences were observed between groups for mortality nor incidence of infectious complications in this study

The usefulness of combined high-frequency percussive ventilation during acute respiratory failure after smoke inhalation.

Inhalation injury and bacterial pneumonia represent some of the most important causes of mortality in burn patients. We describe 11 severely burned patients with acute respiratory failure due to inhalation injury who did not respond adequately to conventional respiratory support. High-frequency percussive ventilation (HFPV) is a recent ventilatory mode, which combines the advantages of conventional ventilation with some of those of high-frequency ventilation. Seven patients developed pulmonary infection during the acute phase; one patient died of multiple organ failure on day 25. All the other patients survived; two developed bronchiolitis obliterans symptoms before discharge. No side-effects were noted and haemodynamic tolerance of HFPV was excellent. Our findings suggest that HFPV can improve pulmonary function and gas exchange in these catastrophic pulmonary failures following inhalation injury

Direct detection and identification of Pseudomonas aeruginosa in clinical samples such as skin biopsy specimens and expectorations by multiplex PCR based on two outer membrane lipoprotein genes, oprI and oprL.

A multiplex PCR test based on the simultaneous amplification of two lipoprotein genes, oprI and oprL, was designed and evaluated for its ability to directly detect fluorescent pseudomonads (amplification of oprI open reading frame, 249 bp) and Pseudomonas aeruginosa (amplification of oprL open reading frame, 504 bp) in clinical material. A collection of reference strains including 20 different species of fluorescent pseudomonads was tested. Positive PCR results for both genes were observed only for P. aeruginosa isolates (n = 150), including strains of clinical and environmental origin, while only one gene, oprI, was amplified from the other fluorescent pseudomonads. All other bacteria tested (n = 15) were negative by the amplification test. The lower detection level for P. aeruginosa was estimated to be 10(2) cells/ml. Preliminary evaluation on testing skin biopsy specimens from patients with burns (n = 14) and sputum samples from cystic fibrosis patients (n = 49) and other patients (n = 19) showed 100% sensitivity and 74% specificity in comparison with culture. This multiplex PCR assay appears promising for the rapid and sensitive detection of P. aeruginosa in clinical specimens. Further evaluation of its specificity in longitudinal clinical studies is warranted