Background. Stromal vascular fraction (SVF) therapy has been performed over the past six years to treat 421 patients by our group in five clinical centers. Autologous SVF, which is a substance containing stem cells, was isolated from lipoaspirate, mixed with platelet-rich plasma (PRP), and administered to patients with degenerative diseases, autoimmune diseases, trauma, aging, and other diseases with unknown etiology. This study aimed to determine the safety of SVF and PRP that were given through infusion, spinal, and intra-articular injection. Methods. The lipoaspirate was treated with a tissue-dissociating enzyme, and then, through centrifugation, SVF was isolated. In addition, blood was drawn from each patient, and PRP was isolated. Autologous PRP and SVF were administered to all subjects by intravenous (IV) injection. A minority group within the population received an additional spinal or intra-articular injection. The type of intervention was determined by each disease evaluation. The cell doses and adverse events for each patient were documented and analyzed. Results. Cell dose that was considered to be safe was less than 10 billion SVF cells in 250 cc of normal saline, for IV injection, and less than 1 billion SVF, for intra-articular and spinal injection. Adverse events were not severe and were treated successfully. Any observed adverse events were identified as a result of spinal or intra-articular injections and were not related to SVF or PRP. Conclusions. Our results showed that administration of high dose of SVF until 10 billion cells in a majority of 421 patients through infusion, spinal, and intra-articular injection was feasible without causing major adverse events and should be further investigated in well-designed phase I-II clinical trial to address the safety and efficacy of therapy.

Background: Platelet-rich plasma (PRP) contains pro-angiogenic growth factors including vascular endothelial growth factor (VEGF). Angiogenesis is a necessary component of wound healing in instances of diabetic foot ulcers (DFU). PRP composition varies depending on methods and donor health status. Our group has developed an improved PRP protocol for diabetes treatment. The aims of this study were to examine the levels of the pro-angiogenic factor VEGF in these patient populations with and without diabetes.Methods: PRP was prepared using 24 mL of whole blood from 13 diabetic and 10 non-diabetic patients registered at Klinik Hayandra. Whole blood in sodium citrate tubes were centrifuged at 1,000 rpm for 5 minutes followed by plasma separation. Plasma samples were centrifuged at 3,000 rpm for 5 minutes.Upper platelet-poor plasma layers were discarded, leaving 5 mL of concentrated platelet containing plasma (PRP). Concentrated plasma samples were mixed, aliquoted, stored at −86 ℃, and pooled for platelet count, VEGF, and total protein analyses. Platelet counting was also performed using fresh whole blood and PRP to measure changes following PRP preparation.Results: Diabetic donors had higher whole blood platelet counts than non-diabetic donors, but this difference was not statistically significant. An average increase of more than 250% in platelet number after PRP preparation using our method was noted in both groups. Freezing-thawing samples at −86 ℃ lysed more than 90% of PRP platelets regardless of diabetes status. Diabetic PRP had lower mean total protein and higher VEGF concentrations. Lysed platelets from diabetic donors released more VEGF than those from non-diabetic donors.Conclusions: PRP from diabetic donors had higher VEGF content making autologous PRP application a promising treatment for DFU. However, this should be investigated another appropriate clinical trial.

Background: Ascorbic acid-2-phosphate has been reported to play a role in cell division and to suppress aging of cell. However, post-thawed cell morphology on various concentration of ascorbic acid is still unclear. In this study, we aimed to observe the morphology of post-thawed adipose-derived stem cells (ADSCs) in medium containing L-ascorbic acid-2-phosphate (LAA2P) (50 and 100 µg/mL).Methods: The cells were isolated from adipose tissue. Isolated cells then cultured and cryopreserved in liquid nitrogen. We detected mRNA expression of type 1 collagen on day 5. Cell seeded in T25 flask using basal medium [Dulbecco's modified Eagle's medium (DMEM) only] as a control group, DMEM with 10% fetal bovine serum (FBS) and antibiotics as DMFA group, while DMFA with ascorbic acid (50 and 100 µg/mL) as ascorbic acid treatment group. Results:The results showed that the cells cultured in DMEM only attached until 96 hours of observation while serum groups with or without ascorbic acid supplementation showed the proliferation until 240 hours of observation. The highest spread size of cell was in a serum group without ascorbic acid supplementation and the highest yield of cells showed in a group with 50 µg/mL of ascorbic acid supplementation. Reduced mRNA expression of type 1 collagen which related to aging was showed in cells cultured without ascorbic acid supplementation.Conclusions: These results showed that ascorbic acid increased the cell division and suppressed the aging processes indicated by normal spread cell in size compared to cell cultured in DMFA without ascorbic acid supplementation.

AbstrakStudi tentang kemampuan adipose-derived stem cells (ADSCs) sebagai sel punca yang dapat berdiferensiasi menjadi kondrosit menggunakan platelet-rich plasma (PRP) sebagai subtitusi fetal bovine serum (FBS) telah banyak dilaporkan. Penggunaan medium pertumbuhan dengan kombinasi ADSCs, PRP dan scaffold sutra masih belum banyak dipelajari dalam rekayasa jaringan kartilago. Studi ini bertujuan untuk mempelajari pengaruh medium yang mengandung 5%, 10% dan 20% PRP terhadap proses kondrogenesis ADSCs manusia yang dikultur pada scaffold sutra Bombyx mori Indonesia. Metode penelitian diawali dengan pembuatan scaffold sutra menggunakan metode salt-leaching, isolasi dan kultur ADSCs manusia dari jaringan lemak, uji pertumbuhan ADSCs pada scaffold sutra dengan variasi konsentrasi PRP pada medium serta analisis kadar glikosaminoglikan (GAG). Hasil penelitian menunjukkan bahwa ADSCs yang dikultur menggunakan PRP lebih tinggi laju pertumbuhannya dibandingkan dikultur menggunakan FBS selama 7 hari pengamatan. Kadar GAG yang disekresikan ADSCs kelompok PRP juga lebih tinggi dibandingkan kelompok FBS. Kadar GAG tertinggi pada hari ke-21 pengamatan adalah medium yang mengandung 20% PRP kemudian 10% dan 5%, sedangkan kadar GAG kelompok kontrol cenderung stabil pada kadar yang rendah. Berdasarkan hasil tersebut, medium yang mengandung PRP memiliki potensi dalam menginduksi kondrogenesis ADSCs yang dikultur pada scaffold sutra.Abstract The studies on adipose-derived stem cells (ADSCs) differentiation into chondrocytes using platelet-rich plasma (PRP) as a substitute for fetal bovine serum (FBS) have been reported. However, the combination of ADSCs, PRP and silk fibroin scaffold has not been widely studied for developing cartilage engineering. Therefore, this research aims to study the effect of medium containing 5%, 10% and 20% PRP towards chondrogenesis of human ADSCs cultured on silk fibroin scaffold from Indonesia Bombyx mori. At first, the silk fibroin scaffold was fabricated using a salt-leaching method, then ADSCs were isolated and cultured from adipose tissues. The assays of growth curve and biocompatibility of silk fibroin scaffold toward ADSCs supplemented by PRP as well as glycosaminoglycans (GAG) concentration were conducted later. The results showed that higher absorbance of proliferation rate was on ADSCs supplemented by various PRP concentrations compare to FBS control group for seven days of observation. Level of GAG, which secreted by ADSCs supplemented by a various concentration of PRP, was also higher than the FBS group. The highest level of GAG on day 21 was observed in 20% PRP group then 10% and 5% PRP, while a group of GAG level is stable at low levels. This study concludes that PRP has the potential to induce chondrogenesis ADSCs which cultured on silk fibroin scaffold.

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