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 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).
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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 lab coats for personal protection should always be worn, and all surfaces should be washed with appropriate biocides. Excess tissue should be disposed of properly as biomedical waste.
1. Materials needed and preparation of the culture solutions
- For cell isolation, expansion, and cryopreservation, obtain the following instruments: sterile scissors; sterile scalpels; sterile tweezers; a 10 cm Petri dish; 15 mL tubes; T25 flasks; a Burker chamber; cryovials; a cell freezing container; an optical microscope with 4x and 10x magnification objectives; and a swinging bucket centrifuge.
- For immunophenotyping, obtain the following instruments and reagents: FACS tubes, sterile phosphate-buffered saline (PBS); Dulbecco's minimal essential medium (DMEM); hPL; dimethyl sulfide; and animal-free trypsin.
- Prepare the complete culture medium by supplementing DMEM with 5% heparin-free hPL. Store the medium at 4 °C for up to 3 weeks, and always use warm (between room temperature and 37 °C). Following GMP guidelines for the production of cell medicines intended for human therapy, do not add any antibiotics to the culture medium.
- Prepare freezing medium containing 20% hPL and 10% dimethyl sulfide in DMEM.
2. Isolation of hASCs from the adipose tissue sample
- Process the adipose tissue sample within 6 h of obtaining it. Before proceeding to the established mechanical xenogeneic-free isolation method, wash the tissue 2x with PBS for 10 min until connective tissue and blood are released.
- Store the obtained adipose tissue sample from abdominoplastic surgery in PBS at 4° C until processing and, in any case, no longer than 24 h, in order to not damage the hASC viability.
- Cut the adipose tissue into smaller pieces of approximately 1 cm2 with sterile scissors.
- With a scalpel, micro-dissect each piece into little fragments with diameters <5 mm, and disperse five of them in a 10 cm Petri dish (Figure 2A).
- In order to attach each fragment, use the scalpel to create incisions on the plastic surface, dragging each fragment until it is firmly stuck to the Petri dish. Use tweezers to help handle the fragment (Figure 2B).
- Gently add 10 mL of complete culture medium in order to cover all the fragments without detaching them.
- Incubate the Petri dishes at 37 °C and 5% CO2. The hASCs will migrate out of the tissue and be selected due to their plastic adherence without enzymatic digestion.
- Until cell confluence, monitor the hASC expansion under an optical microscope at 4x magnification once per day. As the hASCs begin to exit from the tissue, they appear as small clusters around the tissue pieces with a typical fibroblast-like morphology. The hASCs are selected due to their ability to adhere to the plastic surface. Every 72 h, remove as much medium as possible, and replace it with fresh complete medium in order to remove cell debris.
- Leave the fragments of tissue in place until the first passage of cells, which is usually after 7 days.
3. hASC culture after the isolation phase
- When the hASC culture reaches 70%-80% confluency, the hASCs are ready to be detached and further expanded to be either used for other experiments or long-term storage.
- With sterile tweezers, remove and discard all the pieces of tissue from the Petri dish. Remove and discard the exhausted medium, being careful not to touch the hASCs in order not to detach them.
- Carefully wash the cells once with 10 mL of PBS. Add 1 mL of 1x animal-free trypsin, and leave the Petri dish in the incubator at 37 °C for 5 min.
- Check under an optical microscope at 4x magnification if all the cells have detached; otherwise, leave the Petri dish for an additional 2 min in the incubator, and eventually gently tap the plastic surface to support cell detachment.
- Stop the trypsin action by adding 2 mL of complete medium, and collect all the cells in a 15 mL tube.
- In order to remove any remaining trypsin, centrifuge the tube at 300 x g for 5 min. Discard the supernatant, suspend the cells with 5 mL of complete medium, and transfer the cell suspension into a new T25 flask. Keep the hASCs in culture, and expand them until needed.
4. Immunophenotype investigation by flow cytometry
- When the hASC culture reaches 70%-80% confluency, detach the hASCs as previously described in section 3.
- After collecting the cells, suspend them in 1 mL of complete medium, and count an aliquot with the cell counter under an optical microscope at 10x magnification.
- Centrifuge the hASCs at 300 x g for 5 min, and suspend them at 1 x 106 cells/mL of FACS buffer. Transfer 100 µL of the suspension containing 1 x 105 cells into one FACS tube. Use one FACS tube for each investigated antigen, plus one more for each isotypic control.
- Stain the cells with 100 µL of the following antibodies diluted in FACS buffer: anti-CD73 (1:1,000, IgG1, FITC-labeled), anti-CD105 (1:200, IgG1, PE-labeled); anti-CD34 (1:66, IgG1, FITC-labeled); anti-CD45 (1:66, IgG1, FITC-labeled); and isotypic controls for each fluorophore, IgG1 FITC-labeled (1:1,000) and IgG1 PE-labeled (1:50).
- Incubate the samples for 1 h in the dark with agitation at 4 °C. Centrifuge the tubes at 300 x g for 5 min, discard the supernatant, and suspend the cells in 500 µL of FACS buffer.
- Assess the cell immunophenotype with a flow cytometry instrument, recording 50,000 events for each tube inside the gate selected to exclude cell debris. Calculate the percentage of expressing cells as the difference from the specific isotypic control.
5. Proliferation assay of hASC
- When the hASC culture reaches 70%-80% confluency, detach the hASCs as described in section 3.
- After collecting the cells, suspend them in 1 mL of complete medium, and count an aliquot with the cell counter under an optical microscope at 10x magnification.
- Seed 5 x 102 hASCs in 200 µL of complete medium in 96-well transparent plates. Seed the cells in technical replicates, with one well for each time point.
- At 3 days after seeding, start detecting the cell proliferation using a proliferation assay kit following the manufacturer's instructions. In brief, replace the culture medium with 100 µL of fresh medium added to 20 µL of assay reagent per well. Incubate the hASCs for 2.5 h at 37 °C, 5% CO2 for reagent development, and then detect absorbance at 490 nm with a spectrophotometer.
- Express the hASC proliferation as the percentage absorbance after removing the blank absorbance.
6. Cryopreservation and storage of the hASCs
- Detach the cells as described in section 3, and count an aliquot with the cell counter under an optical microscope at 10x magnification.
- Centrifuge the cells at 300 x g for 5 min, and discard the supernatant. Suspend the cells with freezing mix at a density of 1 x 106 hASCs/mL, and transfer 1 mL of the cell solution into one cryovial.
- Put the cryovials at −80 °C in a freezing container that decreases the temperature by 1 °C/min, and then move the cryovials to liquid nitrogen for long-term storage.
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Representative 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 classical spindle-like shape and were smaller and more elongated compared to the control cells in the presence of FBS (Figure 3). The cell immunophenotype evaluated by flow cytometry appeared to be similar between the hASCs cultured with the two supplements. In particular, more than 80%-90% of the hASCs were positive for CD7317 and CD10517, and less than 5% expressed CD3417 and CD4517 (Figure 4). Finally, the proliferation assay revealed a significant increase in cell growth when hPL was added to the medium compared to the FBS control (Figure 5).
Figure 1: Graphical abstract of the protocol. Xenogeneic-free method for the isolation and in vitro expansion of human adipose-derived stem cells. Please click here to view a larger version of this figure.
Figure 2: Adipose tissue cutting and seeding process to obtain hASCs in in vitro culture. (A) Microdissection of the adipose tissue into fragments with diameters <5 mm and dispersion of the fragments in a 10 cm Petri dish. (B) Attachment to the Petri dish after creating incisions on the plastic surface with a scalpel. Please click here to view a larger version of this figure.
Figure 3: Morphology of the hASCs. The hASCs cultured in the xenogeneic-free condition showed a spindle-like shape and elongated morphology. The hASCs cultured in the presence of FBS as a control were bigger and had a bullseye shape. The scale bar represents 100 µm. Please click here to view a larger version of this figure.
Figure 4: Immunophenotype of the hASCs. The immunophenotype of the hASCs was similar in the presence of hPL and FBS. In particular, the hASCs in both conditions could be considered positive for CD73 and CD105 and negative for CD34 and CD45. The error bars indicate the standard error (n = 3). Abbreviations: hPL = human platelet lysate; FBS = fetal bovine serum. Please click here to view a larger version of this figure.
Figure 5: hASC proliferation. The hASCs expanded in xenogeneic-free conditions proliferated significantly more than those in the presence of FBS. ** p < 0.01 (significance evaluated with a Student's t-test, n = 3). The x-axis represents the time in terms of the number of days after seeding. Abbreviations: hPL = human platelet lysate; FBS = fetal bovine serum; hASCs = human adipose-derived stem cells. Please click here to view a larger version of this figure.
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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 properties18,19,20. As a result, hASCs are considered an excellent candidate for cell-based strategies in regenerative medicine21. After isolation from adipose tissue, hASCs require expansion in vitro and, in some cases, should be subjected to a differentiation step before being used for a specific therapeutic application. The entire process, including the isolation, te in vitro expansion, and eventual differentiation, must be conducted in accordance with GMP guidelines to reduce any risks from a translation perspective22,23.
Regarding the isolation process, while the enzymatic digestion and centrifugation steps quickly yield a high number of hASCs in a relatively short time, previous reports have shown that these methods cause possible cell phenotypic changes in terms of cluster differentiation markers compared to other mechanical isolation methods, thus raising questions about the biosafety, cell properties, and behavioral issues when applying these hASCs with human patients3. Moreover, to minimize the risks related to xenogeneic components, recent studies have suggested using synthetic enzymes or mechanical methods to isolate hASCs. However, even synthetic enzymes may possibly modify the cell profile and affect the product quality24. On the contrary, the explant-based method investigated here may be a valid alternative for translational purposes, since the hASCs migrate out of the tissue and adhere to the plastic surfaces naturally, without the use of any xenogeneic reagents9. At the same time, it must be considered that this method evidenced lower cell yield compared to the classic collagenase isolation25.
Regarding the supplement medium, based on recent literature, which agrees that hPL supports hASC expansion to a higher degree than FBS without altering the hASC physiological properties26,27,28,29, we substituted the traditional FBS-added medium with hPL16. Supporting the cell culture with hPL, we overcame the diminished cell yields and the slower recovery related to mechanical isolation. These less-manipulated hASCs proliferated in vitro with a rate permitting the expansion required to reach clinically relevant volumes in an efficient and safe manner30,31. Moreover, the culture with hPL did not alter the stem-like features and immunophenotype typical of hASCs, thus meaning the therapeutic interest of these cells was maintained.
To the best of our knowledge, while a variety of explant isolation protocols have been described for ASCs, our method is the first combining xenogeneic-free mechanical isolation of ASCs with hPL-supplemented medium culture to obtain a valuable cell population for translational applications.
Besides that, the method described in this work has some critical aspects that need to be detailed further. Firstly, the diameters of the minced adipose tissue fragments should not exceed 5 mm to facilitate their adhesion to the plastic surface and allow the migration of the hASCs out of the tissue. Secondly, the presence of adipose tissue fragments in culture together with the hASCs already attached to the plate should be balanced, considering the contamination risk and the need for a sufficient number of migrated cells. Thirdly, researchers need to be aware of a possible reduced growth rate in the first 24 h after the explant seeding; after this period of time, the hPL supplementation stimulates cell recovery and proliferation compared to the classic FBS30.
For the moment, no further steps or modifications have been introduced compared to the original protocol described above.
In conclusion, the complete xenogeneic free method described here provides a simple way of isolating hASCs from adipose tissue, including both lipoaspirates and abdominal adipose tissue, in the absence of animal-derived products, chemical enzymes, or centrifugation steps. Moreover, the hPL-supplemented medium counterbalances the resulting initial cell lower yield and enhances cell proliferation while keeping the therapeutic properties of hASCs unaltered.
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Disclosures
The authors have nothing to disclose.
Acknowledgments
The authors have no acknowledgments.
Materials
| 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 |
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