Standardization and Optimization for the Use of Human Adipose-Derived Stem Cells on an Extracellular Matrix

Abstract

PURPOSE: Extracellular matrix (ECM) use in breast reconstruction surgery has gained popularity with utilization in 56% of tissue-expander based and/or implant-based surgical procedures.1 Combining adipose-derived stem cells (ASC) with an ECM has demonstrated improved tissue incorporation and increased strength of repair in hernia repair.2 Obtaining sufficient ASC number and improving ASC attachment to ECM are two key limiting factors for clinical treatments utilizing ASC on an ECM. With this study, we hope to establish (1)a safe and efficacious population doubling (PD) range under which in vitro expansion doesn’t affect ASC population doubling time (PDT) and/or differentiation capacity (DC); and determine if (2)human platelet rich plasma (PRP) can be used to promote the attachment and proliferation of ASC on an ECM. METHODS: Freshly isolated ASC from 3 individuals were cultured at 4 different seeding densities (n=4) ranging from 500 cells/cm2 to 5000 cells/cm2. Cell numbers were determined at 90% confluence to calculate PDT and PD. Osteogenic, chondrogenic, and adipogenic DC were measured by Alizarin red, Alcian blue, and Oil red O staining, respectively. Freshly isolated PRP from 3 individuals (n=3) or phosphate-buffered saline (PBS) (n=3) was pre-incubated on ECM before seeding with ASC. MTT assays were utilized to assess cell attachment at day 1 and cell proliferation at day 7. RESULTS: ASC were cultured for 9 to 31 days prior to 90% confluence and no significant differences in growth rates were observed among individual specimens and overall for PD 1.6 to 6.2 (n=4, p=0.184)(Fig. 1). The DC over PDs 1.6 to 6.2 also showed no significant changes (osteogenic, n=3, p=0.472; chondrogenic, n=3, p=0.878; adipogenic, n=3, p=0.256). PRP treatment significantly increased cell attachment on ECM (P<0.001) and also significantly increased proliferation compared to the PBS controls across all samples (P<0.001)(Fig. 2).Figure 1: Cell Growth Rate: Population doubling time (PDT) observed among seeding densities 500 cells/cm2 to 5000 cells/cm2 (n=4, p=0.184).Figure 2: Cell Attachment and Proliferation: Significantly increased MTT readings across all groups (PBS:PBS, PRP:PRP, All:All) (n=3, P<0.001). Significantly increased cell presence demonstrated after 7 days (n=3, P<0.001).CONCLUSION: Our results demonstrate that in vitro expansion of ASC within the PD range 1.6 to 6.2 causes no significant change in cell growth rate or regenerative capacity. This suggests in vitro expansion prior to clinical application in this PD range is both safe and effective. Also, PRP treatment was shown to significantly improve ADSC attachment and proliferation on an ECM. We hope that this information will benefit ASC application to ECM in future in vivo studies and clinical applications.