Collagenase-assisted fat dissociation for autologous fat transfer.

BACKGROUND: The quality of fat for autologous transfer procedures has been a major focus of research in the past few years. The primary goal of these efforts is to improve the viability and longevity of the graft in human subjects. One possible factor in the permanence of theses transplants is the size of the adipose tissue grafts. OBJECTIVE: This study evaluated the effects of collagenase digestion on the viability of human adipose tissue. MATERIALS AND METHODS: Samples of fat were obtained from subjects undergoing tumescent liposuction. The tissue was digested in a variety of concentrations of collagenase using optimized methods of processing. The digested fat was also subjected to mock injections through small bore needles. RESULTS: Eight subjects completed the study. The viability of the fat using the optimized methods of collagenase digestion was consistently higher than 79%. During the mock injection trials, the viability of fat was improved from approximately 17% to 84% by collagenase digestion. CONCLUSIONS: Our results show increased viability of human adipose tissue when digested by collagenase. These techniques can be applied to human autologous lipoaugmentation procedures in an effort to improve longevity of the transplanted tissue.

Cryopreservation of human fat for soft tissue augmentation: viability requires use of cryoprotectant and controlled freezing and storage.

BACKGROUND: Autologous fat transfer for soft tissue augmentation has been increasing in recent years. Graft longevity may vary greatly from patient to patient, requiring repeat procedures, often using frozen adipose tissue. Storage usually involves placing syringes of fat directly into a -20 degrees C freezer. However, the viability of fat frozen in this way is controversial. OBJECTIVE: This study tested methods for the optimal storage of adipose tissue harvested by tumescent liposuction. MATERIALS AND METHODS: Aliquots of washed adipose tissue were frozen directly at -20 degrees C or mixed with cryoprotectants, frozen at 1 degree C/min, and subsequently stored in liquid nitrogen vapor phase. Aliquots were subsequently thawed, and adipocyte viability was determined by staining and culture methods. RESULTS: Viability of adipocytes frozen at -20 degrees C was very low when analyzed by staining, and no cultures could be established from any of the specimens. In contrast, viable adipocytes were recovered from samples that were controlled-rate frozen in the presence of cryoprotectants and stored in nitrogen vapor. CONCLUSION. Our results indicate that fat frozen at -20 degrees C is not viable and thus provides no advantage over inert fillers. The methods here described could readily be transferred to the clinical setting after further laboratory study.

Targeting of cells expressing wild-type EGFR and type-III mutant EGFR (EGFRvIII) by anti-EGFR MAb ICR62: a two-pronged attack for tumour therapy.

With a view to their use in cancer therapy, we have produced rat monoclonal antibodies (MAbs) directed against 5 distinct epitopes (A-E) on the external domain of the wild-type human EGF receptor (EGFR). Here, we have investigated the relative binding and anti-tumour activity of our anti-EGFR MAbs against HC2 20d2/c cells, which have been engineered to overexpress the type-III mutated form of the human EGFR (EGFRvIII). We found that anti-EGFR MAbs that are the most effective antagonists of EGFR ligands (e.g., ICR16, ICR62 and ICR80) also bind to cells that overexpress the EGFRvIII. Although these antibodies are potent inhibitors of the growth of cells which express wild-type EGFR, they did not directly inhibit the growth in vitro of EGFRvIII expressing HC2 20d2/c cells, or the constitutive tyrosine kinase activity of this receptor. However, in the presence of human peripheral blood mononuclear cells (PBMC), the rat IgG2b MAb ICR62 induced strong antibody-dependent cell-mediated cytotoxicity (ADCC) against HC2 20d2/c cells in culture. Interestingly, MAb ICR62 also inhibited very effectively experimental lung metastases of HC2 20d2/c cells in athymic nude mice. Our results suggest that anti-EGFR MAb ICR62, which binds to the EGFRvIII, may have potential in the treatment of tumors which overexpress the EGFRvIII via immunological mechanisms such as ADCC. Since tumours that are EGFRvIII positive may also overexpress the wild-type EGFR, the use of anti-EGFR MAbs that target both wild-type and mutant receptors may have advantages over those that target only1form.

Elevated JNK activation contributes to the pathogenesis of human brain tumors.

The ERK pathway is typically associated with activation of the EGF receptor and has been shown to play a major role in promoting several tumor phenotypes. An analogous signaling module, the JNK pathway, has not been shown to be consistently activated by the EGF receptor but is instead more uniformly stimulated by cellular stresses and cytokines. The function of the JNK pathway in primary tumors is unclear as it has been implicated in both promoting apoptosis and cell growth in vitro, which may be a reflection of the cell lines chosen. Primary human brain tumors frequently show overexpression of the EGF receptor. To clarify the role of JNK in tumorigenesis, we have investigated the role of JNK in a large panel of primary human brain tumors and tumor derived cell lines. Here we present evidence that JNK has a major role in promoting tumorigenesis both in vivo and in vitro. Western blot analysis demonstrated that 86% (18 of 21) of primary brain tumors showed evidence of JNK activation but only 38% (8 of 21) showed evidence of ERK activation. Kinase assays revealed that 77% of brain tumor cell lines activated JNK in response to EGF (7 of 13) or had high levels of basal activity (3 of 13), whereas none of six normal cell lines analysed, including astrocytes, had these properties. Of several growth factors examined, EGF produced the highest level of JNK induction in tumor cell lines and the duration of activation was greater than that seen for ERK. Expression of a dominant-negative (dn) form of JNK potently inhibited EGF mediated anchorage independent growth and protection from cell death in two glial tumor cell lines. These findings demonstrate that enhanced JNK activation is frequently found in primary brain tumors and that this activation contributes to phenotypes related to transformation.