Safety assessment of intraportal liver cell application in New Zealand white rabbits under GLP conditions.

Liver cell transplantation (LCT) is considered a new therapeutic strategy for the treatment of acute liver failure and inborn metabolic defects of the liver. Although minimally invasive, known safety risks of the method include portal vein thrombosis and pulmonary embolism. Since no systematic data on these potential side effects exist, we investigated the toxicological profile of repeated intraportal infusion of allogeneic liver cells in 30 rabbits under GLP conditions. Rabbit liver cells were administered once daily for 6 consecutive days at 3 different dose levels, followed by a 2-week recovery period. No test item-related mortality was observed. During cell infusion, clinical findings such as signs of apathy and hyperventilation, moderate elevations of liver enzymes ALT and AST and a slight decrease in AP were observed, all fully reversible. Cell therapy-related macroscopic and histological findings, especially in liver and lungs, were observed in animals of all dose groups. In conclusion, the liver and lungs were identified as potential toxicological target organs of intraportal allogeneic liver cell infusion. A NOAEL (no observed adverse effect level) was not defined because of findings observed also in the low-dose group. No unexpected reactions became apparent in this GLP study. Overall, LCT at total doses up to 12 % (2 % daily over 6 days) of the total liver cell count were tolerated in rabbits. Observed adverse effects are not considered critical for treatment in the intended patient populations provided that a thorough monitoring of safety relevant parameters is in place during the application procedure.

Acute rejection of myofibers in nonhuman primates: key histopathologic features.

The aim of this study was to define the histologic features of acute rejection of myofibers, particularly in the context of therapeutic myogenic cell transplantation. Myoblasts expressing or not expressing ß-galactosidase were transplanted into 13 macaques that were divided into 3 protocols: withdrawal of immunosuppression, low immunosuppression, and progressive reduction of immunosuppression. The biopsy samples were obtained from cell-grafted sites at different intervals, and cryostat sections of biopsies were analyzed. The grafts were lost in all the monkeys at different periods after transplantation depending on the protocol and in association with low blood levels of tacrolimus. In all cases, graft loss was associated with the presence of dense focal accumulations of CD8-positive and CD4-positive lymphocytes and a component of macrophages. The lymphocyte accumulations totally or partially surrounded some myofibers and often invaded them; they were mainly endomysial. These histopathologic patterns in nonhuman primates and their similarity with preliminary observations in humans may facilitate the translation of these results to the histologic diagnosis of acute rejection of myofibers in human clinical trials of myogenic cell transplantation and probably gene therapy.

Immunological tolerance of human hepatocyte xenograft induced by adenovirus vector-mediated CTLA4Ig gene transfer.

BACKGROUND: Systemic administration of CTLA4Ig has been applied in inducing immunological tolerance of hepatocyte implants, but has potential for systemic immune inhibition. This study was designed to induce hepatocyte immunological tolerance by locally expressing CTLA4Ig in an attempt to improve the effectiveness of cell transplantation. METHODS: A normal human liver cell line (L02) was transfected with adenovirus vector containing the CTLA4Ig gene (Ad-CTLA4Ig-EGFP) in vitro, and the expression of CTLA4Ig by transfected cells was assessed by fluorescent imaging and immunocytochemical staining. Transfected cells then were injected into the spleen of Sprague-Dawley rats, the survival of cells was determined by immunohistochemistry, and the immune status was examined through CD4+ and CD69+ T cell-counts and ELISA detection of IL-2 in peripheral blood. RESULTS: L02 cells expressed CTLA4Ig in the cytoplasm for >4 weeks. Surviving L02 cells were observed in the experimental group at 3 and 4 weeks post-transplantation, while none was detected in the control group. Furthermore, the percentages of CD4+ and CD4+CD69+ T cells in the CTLA4-transfected group were 24.5% and 45.1%, markedly lower than those in the control group at 4 weeks post-transplantation (P<0.01). Furthermore, the IL-2 level was also lower in the CTLA4-transfected group than in the control group. CONCLUSION: Adenovirus-mediated CTLA4Ig gene transfer into human hepatocytes has the potential to become an effective method of inducing immunological tolerance in hepatocyte transplantation.

Interaction of olfactory ensheathing cells with other cell types in vitro and after transplantation: glial scars and inflammation.

Olfactory ensheathing cells (OECs) have been investigated extensively as a therapy to promote repair in the injured CNS, with variable efficacy in numerous studies over the previous decade. In many studies that report anatomical and functional recovery, the beneficial effects have been attributed to the ability of OECs to cross the PNS-CNS boundary, their production of growth factors, cell adhesion molecules and extracellular matrix proteins that promote and guide axon growth, and their ability to remyelinate axons. In this brief review, we focus on the interaction between OECs and astrocytes in vivo and in vitro, in the context of how OECs may be overcoming the deleterious effects of the glial scar. Drawing from a selection of different experimental models of spinal injury, we discuss the morphological alterations of the glial scar associated with OEC transplants, and the in vitro research that has begun to elucidate the interaction between OECs and the cell types that compose the glial scar. We also discuss recent research showing that OECs bear properties of immune cells and the consequent implication that they may modulate neuroinflammation when transplanted into CNS injury sites. Future studies in unraveling the molecular interaction between OECs and other glial cells may help explain some of the variability in outcomes when OECs are used as transplants in CNS injury and more importantly, contribute to the optimization of OECs as a cell-based therapy for CNS injury. This article is part of a Special Issue entitled: Understanding olfactory ensheathing glia and their prospect for nervous system repair.

A novel xenograft model of cutaneous T-cell lymphoma.

Cutaneous T-cell lymphomas (CTCLs) are characterized by accumulation of malignant T cells in the skin. Early disease resembles benign skin disorders but during disease progression cutaneous tumors develop, and eventually the malignant T cells can spread to lymph nodes and internal organs. However, because of the lack of suitable animal models, little is known about the mechanisms driving CTCL development and progression in vivo. Here, we describe a novel xenograft model of tumor stage CTCL, where malignant T cells (MyLa2059) are transplanted to NOD/SCID-B2m(-/-) (NOD.Cg-Prkdc(scid) B2m(tm1Unc) /J) mice. Subcutaneous transplantation of the malignant T cells led to rapid tumor formation in 43 of 48 transplantations, whereas transplantation of non-malignant T cells isolated from the same donor did not result in tumor development. Importantly, the tumor growth was significantly suppressed in mice treated with vorinostat when compared to mice treated with vehicle. Furthermore, in most mice the tumors displayed subcutaneous and/or lymphatic dissemination. Histological, immunohistochemical and flow cytometric analyses confirmed that both tumors at the inoculation site, as well as distant subcutaneous and lymphatic tumors, originated from the transplanted malignant T cells. In conclusion, we describe a novel mouse model of tumor stage CTCL for future studies of disease dissemination and preclinical evaluations of new therapeutic strategies.

Neuropathology of olfactory ensheathing cell transplantation into the brain of two amyotrophic lateral sclerosis (ALS) patients.

Although a large number of amyotrophic lateral sclerosis (ALS) patients have undergone transplantation procedures with olfactory ensheathing cells (OECs) in the Bejing Hospital, to our knowledge, no post-mortem neuropathologic analyses have been performed. We examined the post-mortem brain of two Italian patients affected by ALS who underwent cellular transplantation in Beijing with their consent. Our aim was to assess the events following the graft procedure to possibly support the rationale of the treatment strategy. The neuropathologic findings were analyzed on the basis of the limited awareness of the experimental conditions and discussed in relation to the safety, efficacy and long-term outcome of the transplanted cells. Islands of quiescent, undifferentiated cells within the delivery track persisting for up to 12 months-24 months were found. Prominent glial and inflammatory reaction around the delivery track strongly supports the encasement of the graft. Evidence of axonal regeneration, neuronal differentiation and myelination was not seen. The surgical procedure of implantation was not compatible with a neurotrophic effect. The OEC transplantation did not modify the neuropathology of ALS in the two patients. In conclusion, the present neuropathologic analysis does not support a beneficial effect of fetal OEC implantation into the frontal lobes of ALS patients.

In vivo MRI cell tracking: clinical studies.

OBJECTIVE: The purpose of this review is to describe the principles of MRI cell tracking with superparamagnetic iron oxides and the four clinical trials that have been performed. CONCLUSION: Clinical MRI cell tracking is likely to become an important tool at the bedside once (stem) cell therapy becomes mainstream. The most prominent role of this technique probably will be verification of accurate cell delivery with MRI-guided injection, in which interventional radiologists will play a role in the near future. All clinical studies described as of this writing have been performed outside the United States.

Acanthus ilicifolius plant extract prevents DNA alterations in a transplantable Ehrlich ascites carcinoma-bearing murine model.

AIM: To investigate the chemopreventive efficacy of the Indian medicinal plant Acanthus ilicifolius L Acanthaceae in a transplantable Ehrlich ascites carcinoma (EAC)-bearing murine model. METHODS: Male Swiss albino mice were divided into four groups: Group A was the untreated normal control; Group B was the EAC control mice group that received serial, intraperitoneal (ip) inoculations of rapidly proliferating 2 x 10(5) viable EAC cells in 0.2 mL of sterile phosphate buffered saline; Group C was the plant extract-treated group that received the aqueous leaf extract (ALE) of the plant at a dose of 2.5 mg/kg body weight by single ip injections, once daily for 10, 20 and 30 consecutive days following tumour inoculation (ALE control); and Group D was the EAC + ALE-treatment group. The chemopreventive potential of the ALE was evaluated in a murine model by studying various biological parameters and genotoxic markers, such as tumour cell count, mean survival of the animals, haematological indices, hepatocellular histology, immunohistochemical expression of liver metallothionein (MT) protein, sister-chromatid exchanges (SCEs), and DNA alterations. RESULTS: Treatment of the EAC-bearing mice with the ALE significantly (P < 0.001) reduced viable tumour cell count by 68.34% (228.7 x 10(6) +/- 0.53) when compared to EAC control mice (72.4 x 10(6) +/- 0.49), and restored body and organ weights almost to the normal values. ALE administration also increased (P < 0.001) mean survival of the hosts from 35 +/- 3.46 d in EAC control mice to 83 +/- 2.69 d in EAC + ALE-treated mice. Haematological indices also showed marked improvement with administration of ALE in EAC-bearing animals. There was a significant increase in RBC count (P < 0.001), hemoglobin percent (P < 0.001), and haematocrit value (P < 0.001) from 4.3 +/- 0.12, 6.4 +/- 0.93, and 17.63 +/- 0.72 respectively in EAC control mice to 7.1 +/- 0.13, 12.1 +/- 0.77, and 30.23 +/- 0.57 respectively in EAC + ALE-treated group, along with concurrent decrement (P < 0.001) in WBC count from 18.8 +/- 0.54 in EAC control to 8.4 +/- 0.71 in EAC + ALE. Furthermore, treatment with ALE substantially improved hepatocellular architecture and no noticeable neoplastic lesions or foci of cellular alteration were observed. Daily administration of the ALE was found to limit liver MT expression, an important marker of cell proliferation with concomitant reduction in MT immunoreactivity (62.25 +/- 2.58 vs 86.24 +/- 5.69, P < 0.01). ALE was also potentially effective in reducing (P < 0.001) the frequency of SCEs from 14.94 +/- 2.14 in EAC control to 5.12 +/- 1.16 in EAC + ALE-treated group. Finally, in comparison to the EAC control, ALE was able to suppress in vivo DNA damage by abating the generations of 'tailed' DNA by 53.59% (98.65 +/- 2.31 vs 45.06 +/- 1.14, P < 0.001), and DNA single-strand breaks (SSBs) by 38.53% (3.14 +/- 0.31 vs 1.93 +/- 0.23, P < 0.01) in EAC-bearing murine liver. CONCLUSION: Our data indicate that, ALE is beneficial in restoring haematological and hepatic histological profiles and in lengthening the survival of the animals against the proliferation of ascites tumour in vivo. Finally, the chemopreventive efficacy of the ALE is manifested in limiting MT expression and in preventing DNA alterations in murine liver. The promising results of this study suggest further investigation into the chemopreventive mechanisms of the medicinal plant A. ilicifolius in vivo and in vitro.

Hepatocyte transplantation-biology and application.

Transplantation of liver has been remarkably effective in the treatment of liver failure and liver-based inherited metabolic diseases and has revolutionized the care of patients with end-stage liver disease. Unfortunately demand for transplantable livers is progressively outpacing the supply of donated cadaver organs, resulting in longer waiting times and increased mortality for prospective transplant recipients. Hepatocyte transplantation has been proposed as a method to support patients with liver insufficiency. The current knowledge on this method has been review in this article. Now the two-step collagenase perfusion technique is widely used for isolation of hepatocytes. Liver has been considered as an optimal site for hepatocyte transplantation, however, even in this organ the survival rate of transplanted hepatocytes in extremely low. The main obstacle for wider usage of hepatocyte transplantation is their rapid elimination by recipient macrophages. We tried in animal experiments to downregulate the innate process of recipient cellular attack on implanted hepatocytes by irradiation of recipient and elimination of NK cells. Ligation of bile duct and partial hepatectomies facilitated proliferation of accepted donor hepatocytes and formation of bile canaliculi. The described method is now adjusted to clinical conditions.

Electrophysiological maturation and integration of murine fetal cardiomyocytes after transplantation.

In the present study, we investigated the electrophysiological maturation and integration of immature cardiomyocytes after transplantation; maturation and integration are essential to achieve the cardiac regeneration. Murine fetal cardiomyocytes (FCMs) (d12.5-d15.5) expressing enhanced green fluorescent protein under the control of the alpha-actin promoter were injected into cryoinjured areas and adjacent myocardium of cryoinjured mouse ventricles. Viable short axis tissue slices (thickness, 150 microm) of the ventricles were prepared 5 to 6 days after transplantation. Glass microelectrodes were used for measurements of action potentials in transplanted FCMs and host cardiomyocytes within the slices. Stimulation at frequencies of up to 10 Hz was performed via a unipolar electrode placed in viable host tissue. Transplanted FCMs could be distinguished clearly from host tissue by their green fluorescence and their electrophysiological properties: maximal upstroke velocity (V(max)) was significantly lower and action potential duration at 50% repolarization (APD(50)) was significantly longer compared with values of adult cardiomyocytes. Transplanted FCMs surrounded by cryoinjured tissue showed spontaneous electrical and contractile activity, which was in no case synchronous with host tissue. V(max) and APD(50) of these nonintegrated cells matched values of cultivated dissociated FCMs. In contrast, 82% of transplanted FCMs surrounded by viable host tissue were electrically integrated; ie, electrical and contractile activity was synchronous with host tissue and these cells had more mature action potential parameters (significantly higher V(max) and shorter APD(50)) compared with nonintegrated FCMs. In conclusion, electrophysiological maturation and integration of transplanted FCMs depend on an embedment in viable host myocardium. FCMs surrounded by cryoinjured tissue maintain physiological but immature AP properties.

Cellular therapy in Chagas' disease: potential applications in patients with chronic cardiomyopathy.

Nearly a century after its discovery, Chagas' disease, caused by the protozoan Trypanosoma cruzi, remains a major health problem in Latin America. Although efforts in transmission control have contributed to a decrease in the number of new cases, approximately a third of chronic Chagasic individuals have or will develop the symptomatic forms of the disease, mainly cardiomyopathy. Chagas' disease is a progressively debilitating disease, which, at the final stages, there are no currently available treatments other than heart transplantation. In this scenario, cellular therapy is being tested as an alternative for millions of patients with heart dysfunction due to Chagas' disease. In this article, we review the studies of cellular therapy in animal models and in patients with Chagasic cardiomyopathy and the possible mechanisms by which cellular therapy may act in this disease.

Cellular magnetic resonance imaging: current status and future prospects.

Cellular magnetic resonance imaging (CMRI) allows for the tracking of the temporal and spatial migration of cells labeled with MR contrast agents within organs and tissues. This rapidly growing area of experimental research has the potential of translating from bench to bedside and may be used in conjunction with cellular therapy clinical trials or in the evaluation of novel drug therapies. Ex vivo labeling of nonphagocytic cells with superparamagnetic iron oxide nanoparticles or paramagnetic contrast agents (i.e., gadolinium or manganese) allows for the detection of single cells or clusters of labeled cells within target tissues using CMRI following either direct implantation or intravenous injection. However, prior to the translation of experimental cell labeling studies to clinical trials, it is essential to perform preclinical evaluation to demonstrate a lack of toxicity, the ability to scale-up labeling using good manufacturing practice and the ability to detect cells by in vivo MRI in relevant model systems.

A prospective, randomised study comparing two techniques of autologous chondrocyte implantation for osteochondral defects in the knee: Periosteum covered versus type I/III collagen covered.

INTRODUCTION: The results for autologous chondrocyte implantation (ACI) in the treatment of full thickness chondral defects in the knee are encouraging. At present two techniques have been described to retain the chondrocyte suspension within the defect. The first involves using a periosteal cover (ACI-P) and the second involves using a type I/III collagen membrane (ACI-C). To the authors knowledge there are no comparative studies of these two techniques in the current literature. We have therefore undertaken such a study to establish if there is a difference between the 2 techniques based on a clinical and arthroscopic assessment. METHODS: A total of 68 patients with a mean age of 30.52 years with symptomatic articular cartilage defects were randomised to have either ACI-P (33 patients) or ACI-C (35 patients). The mean defect size was 4.54 cm2. All patients were followed up at 24 months. RESULTS: A clinical and functional assessment showed that 74% of patients had a good or excellent result following the ACI-C compared with 67% after the ACI-P at 2 years. Arthroscopy at 1 year also demonstrated similar results for both techniques. However, 36.4% of the ACI-P grafts required shaving for hypertrophy compared with none for the ACI-C grafts at 1 year. DISCUSSION: This study has shown no statistical difference between the clinical outcome of ACI-C versus ACI-P at 2 years. A significant number of patients who had the ACI-P required shaving of a hypertrophied graft. We conclude that there is no advantage in using periosteum as a cover for retaining chondrocytes within an osteochondral defect; as a result we advocate the use of an alternative cover such as a manufactured type I/III collagen membrane.

Spinal glial activation in a new rat model of bone cancer pain produced by prostate cancer cell inoculation of the tibia.

Studies suggest that astrocytes and microglia in the spinal cord are involved in the development of persistent pain induced by tissue inflammation and nerve injury. However, the role of glial cells in bone cancer pain is not well understood. The present study evaluated the spinal glial activation in a novel rat model of bone cancer pain produced by injecting AT-3.1 prostate cancer cells into the unilateral tibia of male Copenhagen rats. The structural damage to the tibia was monitored by radiological analysis. The thermal hyperalgesia, mechanical hyperalgesia and allodynia, and spontaneous flinch were measured. The results showed that: (1) inoculation of prostate cancer cells, but not the vehicle Hank's solution, induced progressive bone destruction at the proximal epiphysis of the tibia from day 7-20 post inoculation; (2) the inoculation also induced progressive thermal hyperalgesia, mechanical hyperalgesia, mechanical allodynia, and spontaneous flinches; (3) astrocytes and microglia were significantly activated in the spinal cord ipsilateral to the cancer leg, characterized by enhanced immunostaining of both glial fibrillary acidic protein (GFAP, astrocyte marker) and OX-42 (microglial marker); (4) IL-1beta was up-regulated in the ipsilateral spinal cord, evidenced by an increase of IL-1beta immunostained astrocytes. These results demonstrate that injection of AT-3.1 prostate cancer cells into the tibia produces progressive hyperalgesia and allodynia associated with the progression of tibia destruction, indicating the successful establishment of a novel male rat model of bone cancer pain. Further, bone cancer activates spinal glial cells, which may release IL-1beta and other cytokines and contribute to hyperalgesia.

Iron oxide nanoparticle-labeled rat smooth muscle cells: cardiac MR imaging for cell graft monitoring and quantitation.

PURPOSE: To perform a quantitative analysis of anionic maghemite nanoparticle-labeled cells in vitro and determine the effect of labeling on signal intensity at magnetic resonance (MR) imaging. MATERIALS AND METHODS: The study was approved by the institutional animal care and use committee at Hôpital Bichat. In vitro cell proliferation, iron content per cell, and MR signal intensity of cells were measured in agarose phantoms for 0-14 days of culture after labeling of rat smooth muscle cells with anionic maghemite nanoparticles. Next, iron oxide-labeled smooth muscle cells were injected into healthy hearts and hearts with ischemic injury in seven live Fisher rats. Ex vivo MR imaging experiments in excised hearts 2 and 48 hours after injection were performed with a 1.5-T medical imaging system by using T2-weighted gradient-echo and spin-echo sequences. Histologic sections were obtained after MR imaging. Correlation analyses between division factor of iron load and cell amplification factor and between 1/T2 and number of labeled cells or number of days in culture were performed by using linear regression. RESULTS: Viability of smooth muscle cells was not affected by magnetic labeling. Transmission electron micrographs of cells revealed the presence of iron oxide nanoparticles in vesicles up to day 14 of culture. Intracellular iron concentration decreased in parallel with cell division (r2 = 0.99) and was correlated with MR signal intensity (r2 = 0.95). T2*-weighted MR images of excised rat hearts showed hypointense signal in myocardium at 2 and 48 hours after local injection of labeled cells. Subsequent histologic staining evidenced iron oxide nanoparticles within cells and confirmed the presence of the original cells at 2 and 48 hours after implantation. CONCLUSION: Magnetic labeling of smooth muscle cells with anionic maghemite nanoparticles allows detection of cells with MR imaging after local transplantation in the heart.

[Transplantation of retinal pigment pithelium (RPE) following CNV removal in patients with AMD. Techniques, results, outlook]. / Transplantation von retinalem Pigmentepithel (RPE) nach CNV-Exzision bei altersabhängiger Makuladegeneration. Techniken, Ergebnisse und Perspektiven.

Neovascular age-related macular degeneration (AMD) has become the leading cause for severe visual loss in all industrialized nations. Surgical excision of choroidal neovascularizations (CNV) is technically feasible but invariably associated with inadvertent removal of corresponding retinal pigment epithelium (RPE) and subsequent atrophy of the choriocapillaris, with the latter two layers being a prerequisite for normal photoreceptor function. To cover the RPE defect both heterologous and homologous RPE cell suspensions have been injected into the subretinal space. The lack of functional improvement has been attributed to various factors including RPE cell dedifferentiation, failure of adherence to Bruch's membrane as well as development of a regular RPE cell monolayer. Therefore, techniques for translocating intact autologous RPE cell sheets have been sought and preservation of foveal neurosensory functions has recently been successfully demonstrated. Besides translocation of a full-thickness RPE/Bruch's membrane/choroid patch outside the macular area, superfluous choroidal tissue may be ablated intraocularly using an excimer laser prior to translocation. Besides recent pharmacological approaches including anti-VEGF agents, these surgical developments open new perspectives for patients with neovascular AMD.

New concepts and tools in imaging for the study of neurodegenerative disease.

Existing technologies permit the detection of changes in neurotransmitter and/or neuroreceptor expression. This may be useful for diagnosis, for monitoring disease progression, and for assessing the pathogenesis of complications associated with long-term treatment. Although the binding of [11C]raclopride to D2 receptors is subject to competition from endogenous dopamine, this can be exploited to estimate changes in synaptic levels of dopamine. Assessment of processes downstream to the receptor will require the development of new approaches.

[Isolation, culture and intraspleenic transplantation of rat hepatic oval cells].

OBJECTIVE: To observe the evolution and differentiation of hepatic oval cells after transplanted into the spleens of homogenous rats, providing experimental data for treating hepatic failure with hepatic stem cells. METHODS: A two-step perfusion procedure was used to separate hepatic parenchymal cells from nonparenchymal cells. Then the suspension of nonparenchymal cells was centrifuged in Percoll gradients. The isolated cells were cultured, identified, and then transplanted into the spleens of homogenous rats undergone 2/3 hepatectomy. RESULTS: The obtained cells were various in size with ovoid nuclei and inadequate cytoplasm. After 12 hours' culture, they revealed the characteristics of epithelial cells. Both the freshly isolated and cultured cells showed positive staining for cytokeratin 19 (CK19), OV6, alpha fetal protein (AFP), but negative for leucocyte common antigen (LCA). After intraspleenic transplantation into homogenous rats undergone partial hepatectomy, hepatic oval cells were differentiated into liver tissue-like structure including hepatocyte cords and bile ducts, and formed hepaticized spleen. But this kind of structure was not observed in the controls. CONCLUSION: The isolated rat hepatic oval cells show the biological characteristics of hepatic stem cells and can differentiate into hepatocytes and biliary epithelial cells under appropriate circumstances.

Measurement in vivo of the survival rate in autologous adipocyte transplantation.

Up until now, research on fat cells has been unable to prove their survival rate objectively in vivo. In this article, the first application of the cell surface marker PKH26 in the fat cells of rats is reported. In a study of 48 Lewis rats, this method enabled the objective stereometry of viable and necrotic grafts after variable follow-up times in groups of eight animals each. The best survival rate was 30.41 percent, and the best implantation site was the interscapular subcutis. During follow-up, a characteristic change in size of the viable fat cells matched the in vitro findings of various investigators. Because of the surface marking, it could be proved that the viable cells found after 6 months were transplanted cells that had undergone a cycle of fat deprivation and regaining. This is proof of the cell survival theory postulated by Peer in 1950.