Abstract
The efficacy and economy of most in vitro human models used in research is limited by the lack of a physiologically-relevant three-dimensional perfused environment and the inability to noninvasively quantify the structural and biochemical characteristics of the tissue. The goal of this project was to develop a perfusion bioreactor system compatible with two-photon imaging to noninvasively assess tissue engineered human adipose tissue structure and function in vitro. Three-dimensional (3D) vascularized human adipose tissues were engineered in vitro, before being introduced to a perfusion environment and tracked over time by automated quantification of endogenous markers of metabolism using two-photon excited fluorescence (TPEF). Depth-resolved image stacks were analyzed for redox ratio metabolic profiling and compared to prior analyses performed on 3D engineered adipose tissue in static culture. Traditional assessments with H&E staining were used to qualitatively measure extracellular matrix generation and cell density with respect to location within the tissue. The distribution of cells within the tissue and average cellular redox ratios were different between static and perfusion cultures, while the trends of decreased redox ratio and increased cellular proliferation with time in both static and perfusion cultures were similar. These results establish a basis for noninvasive optical tracking of tissue structure and function in vitro, which can be applied to future studies to assess tissue development or drug toxicity screening and disease progression.
Highlights
Two-dimensional (2D) cell culture systems and animal models are routinely used to assess toxicity for drug screening and to gain insight into diseases
Ethics Statement The protocols for engineering 3D vascularized human adipose tissue in vitro have been described previously by our lab [15]. human adipose derived stem cells (hASCs) were acquired from subcutaneous adipose tissue which was obtained from abdominoplasties approved under Tufts University Institutional Review Board (IRB) (Tufts University IRB Protocol #0906007) from Tufts Medical Center, Department of Plastic Surgery
The average cellular redox ratios gathered through two-photon excited fluorescence (TPEF) imaging and subsequent image processing showed similar trends as has been observed in static cultures; consistent redox ratios over the first week were followed by a significant drop at 2 wks
Summary
Two-dimensional (2D) cell culture systems and animal models are routinely used to assess toxicity for drug screening and to gain insight into diseases. Animal models are utilized for many studies; when assessed for complex disease treatments including but not limited to treatments for viral (HIV), multibacillary (leprosy), and respiratory diseases, they usually fail to adequately represent how clinical treatments affect humans [2,3,4]. This is due to the complex differences in immunological responses that humans and other animals exhibit compared to one another, as well as differences in metabolic and physiological responses. The ability to combine cells and matrices to generate 3D human tissues in bioreactors has advanced rapidly in recent years to make tissue engineered constructs utilizing human cell sources a more advantageous model for studies relevant to clinical applications
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