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An Automated Walkaway System to Perform Differentiation of 3D Mesenchymal Stem Cell Spheroids

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February 13, 2017

Authors: Brad Larson, BioTek Instruments, Inc; Glauco R. Souza, Nano3D Biosciences, Inc.; Jan Seldin, Greiner Bio-One, Inc.

Nano3D     Greiner

Introduction

Human mesenchymal stem cells (hMSCs) are multipotent and found in multiple areas of the body including bone marrow, skeletal muscle, dermis, and blood. The cells are known for their ease of isolation and ability to differentiate and mature into multiple lineages including adipocytes, chondrocytes, and osteocytes. hMSCs also play a critical role in adult tissue repair, therefore are of great interest in tissue engineering applications. For example, as adult cartilage cannot repair itself, chondrocyte-derived hMSCs may be used for cartilage repair applications, and in fact, transplantation of spheroidal chondrocytes is already being studied as a treatment for hip joint cartilage defects. Initial hMSC experimentation involved two-dimensional (2D) cell culture in a monolayer. However, culturing the cells in this manner results in a loss of replicative ability, and differentiation capability over time. A number of techniques to culture hMSCs in a threedimensional (3D) format were then incorporated, such as pellet and micromass culture. These methods better exemplified the differentiation process, but disadvantages included requiring large numbers of cells, difficult manual processing steps, and a high overall cost per method. Recently developed 3D cell culture technologies, which have the ability to create spheroids of smaller cell numbers in high density microplates, can overcome the limitations of earlier methods while still providing the necessary environment for proper stem cell differentiation.

Complete differentiation from multipotent hMSCs to final target lineages, such as chondrocytes, typically takes 14-28 days. With media exchanges required every 2-3 days, a manual process is not only tedious, but when working with nonattached cells, increases the risk of accidental spheroid removal. Automating the processing steps and incorporating a 3D magnetic bioprinting method, such as the 384-Well BiO Assay Kit and NanoShuttle-PL particles from nano3D Biosciences, frees researchers to perform other tasks and increases repeatability with little to no risk of spheroid loss.

Here we demonstrate the validation of a combined solution to perform automated chondrocyte differentiation from 3D hMSC spheroids, where all instrumentation was contained within a laminar flow hood. A combination washer/dispenser with magnetic plate adapter was used for media exchanges, while an automated incubator maintained proper microplate environmental conditions between exchanges. Label-free cellular imaging was performed following media exchanges to confirm maintenance of spheroids during processing. Immunofluorescence following differentiation confirmed the effectiveness of the system for use with critical stem cell differentiation.

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