Amniotic growth factors enhanced human pre‐adipocyte cell viability and differentiation under hypoxia

Abstract

One of the major drawbacks associated with autologous fat grafting is unpredictable graft retention. Various efforts to improve the survivability of these cells have been explored, but these methods are time-consuming, complex, and demand significant technical skill.In our study, we examine the use of cryopreserved amniotic membrane as a source of exogenous growth factors to improve adipocyte survivability under normal and hypoxic conditions. Human primary preadipocytes were cultured in a gelatin-ferulic acid (Gtn-FA) hydrogel with variable oxygen concentration and treated with amniotic membranederived condition medium (CM) for 7 days. This hydrogel provides a hypoxic environment and also creates a 3D cell culture to better mimic recipient site conditions. The O 2 concentration in the hydrogel was measured by electron paramagnetic resonance oxygen imaging (EPROI). The conjugation of FA was confirmed by FTIR and NMR spectroscopy.The cell viability and adipocyte differentiation were analyzed by alamarBlue™ assay, Oil Red O staining, and RT-qPCR. The expression of genes: Pref-1, C/EBP β, C/EBP α, PPAR-ƴ, SLC2A4, and VEGF-A were quantified. The cell viability results show that the 50% CM showed significantly higher cell pre-adipocyte cell viability. In addition, compared to normal conditions, hypoxia/CM provided higher PPAR-ƴ (p < .05), SLC2A4, and VEGF-A (p < .05) (early and terminal differentiating markers) mRNA expression. This finding demonstrates the efficacy of amniotic CM supplementation as a novel way to promote adipocyte survival and retention via the expression of key gene markers for differentiation and angiogenesis.

Nondestructive, longitudinal, 3D oxygen imaging of cells in a multi-well plate using pulse electron paramagnetic resonance imaging

“…For the past two decades, EPROI has been primarily used for cancer hypoxia research with few notable results, such as the demonstration of oxygen-guided radiation therapy in three different tumor models and rabbit VX-2 tumor pO 2 imaging using a human-sized EPROI instrument [38][39][40][41][42][43] . Recently, the applications of EPROI have been extended to the assessment of biomaterials, engineered grafts, and cell encapsulation devices 13,14,24,30,[44][45][46][47][48] .…”

In vitro oxygen imaging of acellular and cell-loaded beta cell replacement devices

“…Figure 2 shows the data from trityl toxicity studies using four different cell types: fibroblast and insulin producing cells (beta-TC6, MIN6, and human islets) for 1 h, 24 h, and 48 h periods. The typical concentration of trityl used for in vitro pO 2 imaging is 1 mM or less throughout this and other studies in the field 34 , 38 , 70 , 71 . We performed the toxicity study using eight concentrations from 0 (control) to 8 mM.…”

Ojeok-san enhances platinum sensitivity in ovarian cancer by regulating adipocyte paracrine IGF1 secretion

“…First of all, oil red O staining experiments showed that HPAd cells gradually increased lipid droplets with the increase of induction time, with slightly more lipid droplets on day 7 compared to day 1 and more on day 14 (Supplementary Figure 1A). Further, qRT-PCR was used to detect adipogenic differentiation markers [ 23 ]. Compared with day 1 and day 7, the expression of Pref-1 was significantly decreased (0.01 ± 0.00, p < 0.05), and the expression of C/EBPβ, PPARγ, SLC2A4, C/EBPα was significantly increased (2.62 ± 0.09, 48.80 ± 0.67, 343.78 ± 24.75, 58.09 ± 12.35, p < 0.05) (Supplementary Figure 1b ).…”

“…The qRT-PCR experiment was performed with reference to other people’s research [ 23 ]. Briefly, HPAd cells on day 1 and day 7 were collected and lysed by TRIzol reagent (Invitrogen, USA) to obtain total RNA.…”