An injectable non-cross-linked hyaluronic-acid gel containing therapeutic spheroids of human adipose-derived stem cells

Koji Kanayama,Rintaro Asahi,Kentaro Doi,Shinichiro Kuno,Kotaro Yoshimura,Takanobu Mashiko,Jingwei Feng,Kazuhide Mineda,Ataru Sunaga,Szu-Hsien Wu,Kahori Kinoshita

doi:10.1038/s41598-017-01528-3

Koji Kanayama, Rintaro Asahi + Show 9 more

Open Access

https://doi.org/10.1038/s41598-017-01528-3

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Abstract

For chronic wounds, the delivery of stem cells in spheroidal structures can enhance graft survival and stem cell potency. We describe an easy method for the 3D culture of adipose-derived stem/stromal cells (ASCs) to prepare a ready-to-use injectable. We transferred suspensions of monolayer-cultured ASCs to a syringe containing hyaluronic acid (HA) gel, and then incubated the syringe as a 3D culture vessel. Spheroids of cells formed after 12 h. We found that 6 × 106 ASCs/ml in 3% HA gel achieved the highest spheroid density with appropriate spheroid sizes (20–100 µm). Immunocytology revealed that the stem cell markers, NANOG, OCT3/4, SOX-2, and SSEA-3 were up-regulated in the ASC spheroids compared with those in nonadherent-dish spheroids or in monolayer cultured ASCs. In delayed wound healing mice models, diabetic ulcers treated with ASC spheroids demonstrated faster wound epithelialization with thicker dermis than those treated with vehicle alone or monolayer cultured ASCs. In irradiated skin ulcers in immunodeficient mice, ASC spheroids exhibited faster healing and outstanding angiogenic potential partly by direct differentiation into α-SMA+ pericytes. Our method of 3D in-syringe HA gel culture produced clinically relevant amounts of ready-to-inject human ASC microspheroids that exhibited superior stemness in vitro and therapeutic efficacy in pathological wound repair in vivo.

Highlights

About 1% of the population suffers from some form of chronic wound at any given time[1]
Using a syringe as a 3D culture vessel, we developed a new method to make adipose-derived stem/stromal cells (ASCs) spheroids in a syringe filled with Hyaluronic acid (HA) gel (HA ASC spheroids)
In HA ASC spheroids, we found a NANOG-negative surface zone and a NANOG-positive core zone of spheroids (Fig. 3 A-middle), suggesting NANOG expression may correlate with oxygen level created by innate structure of spheroid

Summary

Introduction

About 1% of the population suffers from some form of chronic wound at any given time[1]. In model systems and regenerative therapy, the ASC culture evolves from a monolayer culture to sheet, spheroid, organoid, and, large multiplexed systems, mimicking the body’s organization from cells to layered tissues or simple organs[7]. On the other hand, evolved three dimensional (3D) culture systems help to build sophisticated multicellular constructs with extracellular matrix (ECM) and demonstrate better therapeutic efficacy[9, 10], leading to a new trend in stem cell therapy[11,12,13]. The method is very simple and productive and requires no special effort to extract the spheroids from the HA gel for therapies. We performed in vivo experiments using chronic-wound models to test the therapeutic efficacy and elucidate how the ASC spheroids facilitate wound healing

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