Isolation and Culture of Human Adipose-Derived Stem Cells With an Innovative Xenogeneic-Free Method for Human Therapy

Summary

Xenogeneic (chemical or animal-derived) products introduced in the cell therapy preparation/manipulation steps are associated with an increased risk of immune reactivity and pathogenic transmission in host patients. Here, a complete xenogeneic-free method for the isolation and in vitro expansion of human adipose-derived stem cells is described.

Abstract

Considering the increasing impact of stem cell therapy, biosafety concerns have been raised regarding potential contamination or infection transmission due to the introduction of animal-derived products during in vitro manipulation. The xenogeneic components, such as collagenase or fetal bovine serum, commonly used during the cell isolation and expansion steps could be associated with the potential risks of immune reactivity or viral, bacterial, and prion infection in the receiving patients. Following good manufacturing practice guidelines, chemical tissue dissociation should be avoided, while fetal bovine serum (FBS) can be substituted with xenogeneic-free supplements. Moreover, to ensure the safety of cell products, the definition of more reliable and reproducible methods is important. We have developed an innovative, completely xenogeneic-free method for the isolation and in vitro expansion of human adipose-derived stem cells without altering their properties compared to collagenase FBS-cultured standard protocols. Here, human adipose-derived stem cells (hASCs) were isolated from abdominal adipose tissue. The sample was mechanically minced with scissors/a scalpel, micro-dissected and mechanically dispersed in a 10 cm Petri dish, and prepared with scalpel incisions to facilitate the attachment of the tissue fragments and the migration of hASCs. Following the washing steps, hASCs were selected due to their plastic adherence without enzymatic digestion. The isolated hASCs were cultured with medium supplemented with 5% heparin-free human platelet lysate and detached with an animal-free trypsin substitute. Following good manufacturing practice (GMP) directions on the production of cell products intended for human therapy, no antibiotics were used in any culture media.

Introduction

In the last decades, the increasing demand for innovative therapeutic treatments has given rise to significant efforts and resource investment in the translational medicine field¹. Cell-based products are associated with risks determined by the cell source, the manufacturing process (isolation, expansion, or genetic modification), and the non-cellular supplements (enzymes, growth factors, culture supplements, and antibiotics), and these risk factors depend on the specific therapeutic indication. The quality, safety, and efficacy of the final product could be deeply influenced by the above-indicated elements². Stem cell t....

Protocol

hASCs were isolated from the abdominal adipose tissue of a healthy woman who underwent breast reconstruction using abdominal autologous flaps (deep inferior epigastric perforator flaps, [DIEP]) at the University Hospital of Lausanne, CHUV, Lausanne, Switzerland. The discarded part of the flap and the adipose tissue was obtained after the patient signed informed consent. All protocols were reviewed and approved by the hospital's Biobank Department DAL (number 314 GGC) and ethics committees in accordance with the Declaration of Helsinki.

NOTE: All steps must be performed under a laminar flow hood and with aseptic conditions. Gloves and la....

Results

Applying the isolation method detailed above, hASCs were successfully obtained from abdominal adipose tissue samples without the use of collagenase. Moreover, the hASCs were expanded in complete xenogeneic-free conditions in the presence of hPL and without any other components of animal origin. The following results support the protocol and are obtained from hASCs cultured in parallel with hPL and with FBS as the control condition.

After the initial cluster appearance, the hASCs showed the cla.......

Discussion

Adipose-derived stem cells have attracted the interest of translational research in the last decade due to their abundance, quick and affordable isolation methods, high in vitro/in vivo proliferation rate, and stemness/differentiation properties^18,19,20. As a result, hASCs are considered an excellent candidate for cell-based strategies in regenerative medicine²¹. After isolation from adi.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
15 mL tubes	euroclone	et5015b
anti-CD105	BD Biosciences	BD560839
anti-CD34	BD Biosciences	BD555821
anti-CD45	BD Biosciences	BD555482
anti-CD73	BD Biosciences	BD561254
autoMACS Rinsing Solution (FACS buffer)	Miltenyi	130-091-222
BD Accuri C6 apparatus (flow cytometry instrument)	BD accuri	-
Burker chamber	Blaubrand	717810
Cell freezing container	corning	CLS432002
CellTiter 96 AQueous One Solution Cell Proliferation Assay	Promega	G3582
CoolCell Freezing container	Corning	CLS432002
Cryovials	clearline	390701
Dimethyl sulfide	Sigma Aldrich	D2650-100mL
disposabile blade scalpel	paragon	bs 2982
Dulbecco's Modified Eagle's Medium - high glucose	GIBCO	11965092
Human Platelet Lysate FD (GMP grade)	Stemulate	PL-NH-500
Infinite F50 spectrophotometer	Tecan	-
Optical microscope with 4x and 10x magnification objectives	Olympus	CKX41
Petri dish 10 cm	Greiner bio-one	664160
Sterile scalpels	Reda	07104-00
Sterile scissors	Bochem	4071
Sterile tweezers	Bochem	1152
Swinging bucket centrifuge	Sigma	3-16K
T25 flasks	Greiner bio-one	6910170
TrypLe (animal free trypsin substitute)	GIBCO	12604-013