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.
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.
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 therapy requires adherence to biosafety principles; the potential risks of pathogenic transmission with animal-derived products in cell culture could be problematic, and the thorough testing of any product introduced in the manufacturing is essential3.
The traditional method to isolate human adipose-derived stem cells (hASCs) involves an enzymatic digestion performed with collagenase followed by washing steps through centrifugation4. While enzymatic isolation is generally considered more efficient than other mechanical techniques in terms of cell yields and viability, the animal-derived components used, such as collagenase, are considered more than minimally manipulated by the U.S. Food and Drug Administration. This means there is a significantly increased risk of immune reactions or disease transmission, thus limiting the translation of hASC therapy to clinical settings5,6.
Trypsin-based digestion is another enzymatic protocol to isolate ASCs. Different techniques have been described with slight modifications in terms of the trypsin concentration, centrifugation speed, and incubation time. Unfortunately, this method is not well described, and a lack of comparison exists in the literature, particularly with the mechanical isolation protocols7. However, in terms of the translatability of the approach, trypsin has the same drawbacks of collagenase.
Alternative isolation methods to obtain ASCs, based on mechanical forces and without enzyme addition, involve high-speed centrifugation (800 x g or 1,280 x g, 15 min) of the adipose tissue fragments. Then, the pellet is incubated with a red blood cell lysis buffer (5 min), followed by another centrifugation step at 600 x g before resuspension in culture medium. Despite a greater number of cells being isolated in the first days compared to the explant methods, a previous study showed lower or absent proliferation beyond the second week of culture8.
Besides that, further manipulation with xenogeneic added medium, such as fetal bovine serum (FBS), which is used as a growth factor supplement for cell culture, is associated with an increased risk of immune reactivity and exposure to viral, bacterial, or prion infections of the host patient9,10. Immune reactions and urticariform rash development have been already described in individuals receiving several doses of mesenchymal stem cells produced with FBS11. Furthermore, FBS is subjected to batch-to-batch variability, which can have an impact on the final product quality12.
In accordance with good manufacturing practice (GMP) guidelines, enzymatic tissue dissociation should be avoided, and FBS should be substituted with xenogeneic-free supplements. These steps, together with more reliable and reproducible protocols, are essential to support the application of cell therapy3,13.
In this context, human platelet lysate (hPL) has been suggested as a substitute for FBS since it is a cell-free, protein-containing, growth factor-enriched supplement, and it was earlier introduced among clinical-grade cell-based products as an additive of growth medium for in vitro cell culture and expansion14,15. As hPL is a human-derived product, it is frequently used as a substitute for FBS during the in vitro culture of hASCs intended for clinical applications, thus reducing issues regarding immunological reactions and infections related to FBS translatability15,16.
Despite the higher production costs, it has already been demonstrated that compared to FBS, hPL supports cell viability for many cell types, increases proliferation, delays senescence, assures genomic stability, and conserves the cellular immunophenotype even in late cell passages; all these elements support the switching toward this culture supplement11.
The aim of this work was to develop a standardized protocol to isolate and culture hASCs with a complete animal-free method, without modifying the cell physiology and stemness properties in comparison to classical FBS-cultured hASCs (Figure 1).
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 properties18,19,20. As a result, hASCs are considered an excellent candidate for cell-based strategies in regenerative medicine21. After isolation from adi…
The authors have nothing to disclose.
The authors have no acknowledgments.
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 |