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1. Introduction
Bone marrow stromal cells (BMSCs) have extensively been tested at the preclinical and clinical levels for the treatment of complex bone-related injuries, such as nonunion [1–4], avascular osteonecrosis [5, 6], critical-sized defects [1, 7–12], and osteochondral lesions [13–19] because of their inherent potential to differentiate into multiple skeletal cell types [20–22], modulate inflammatory responses [23–28], and support angiogenesis [29–32].
The treatment of these conditions requires the correct combination of biological (cells and scaffolds) and mechanical factors [33–35]. To replace bone autografts—the current gold standard—in the biological component, BMSCs must be expanded in vitro on a large scale by using good manufacturing practice (GMP) [36–45]. Although a wide variety of methods have been reported to manufacture GMP-grade BMSCs, a still major challenge for the generation of BMSC products is to scale up the processes while maintaining critical cell phenotypic and functional characteristics [25, 26]. Until now, there is no consensus as to which reagents, cell culture medium, and culture systems should be used and which tests should be performed to ensure the safety and efficacy of the final product [27–29].
Therefore, for the successful translation of BMSC potential to the clinic, it is imperative to develop standard procedures for cell production, which, in addition to being evidence-based, well-documented, cost-effective, clinically practical, and incorporating GMP, also guarantee the preservation of BMSC potency [46, 47]. As one of the main orthopedic centers in Brazil, we have established an in-house facility for the isolation and large-scale expansion of functionally certified clinical-grade BMSCs. Here, we report our general procedures, which comply both with GMP standards and the Brazilian regulatory rules for cell manufacturing for therapeutic purposes. These procedures will serve as the basis for BMSC production for future applications in our center, aiming at bone repair.
2. Materials and Methods
2.1. Reagents and Materials
All reagents and materials used for BMSC isolation, expansion, and cryopreservation were certified to be of clinical grade. None of these reagents were aliquoted. To assure traceability, the lot and/or serial numbers of all reagents and materials used in each assay were registered. Reagents used in in vitro differentiation assays and immunophenotyping, when not of clinical grade, were certified to have been produced under good laboratory practices and had defined chemical purity standards. A complete list of all reagents used with all related information is available in Supplementary Materials (Supplementary Table 1). The technical procedures described herein were adapted to comply with the rules of the Brazilian Health Regulatory Agency (ANVISA, Collegiate Board Resolution—RDC Nos. 09/2011 and 214/2018) for the development of cell products intended for use in humans.
2.2. Sample Donor Eligibility Criteria
The study design, the procedures used for its execution, and sample collection were approved by the institutional ethics committee (No. 41660415.3.0000.5273). Subjects of both sexes who were older than 18 years old with indications for total hip replacement were invited to donate the discarded acetabular bone/marrow. After providing written informed consent to donate the samples, a brief questionnaire was used to assess the risk of blood-borne infections. The donors were also questioned about previously diagnosed comorbidities, such as rheumatoid arthritis, diabetes mellitus, bone marrow dysplasia, and malignant tumors, the use of immunosuppressant drugs, and any history of drug and/or alcohol abuse. If any of these were disclosed, the donor was excluded from the study. To those selected, further inclusion criteria were applied: a
Table 1
Characteristics of donor patients and colony-forming efficiency (CFE) results.
Gender | Age | TNC (105) | CFU-F/105 | Colony diameter (mm) | |
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BMSC 01 | M | 54 | 620 | 7.66 | 9.1 |
BMSC 02 | F | 61 | 520 | 8.33 | 8.4 |
BMSC 03 | F | 54 | 600 | 19.33 | 5.0 |
BMSC 04 | M | 48 | 590 | 17.33 | 4.8 |
BMSC 05 | F | 55 | 350 | 13.5 | 4.0 |
BMSC 06 | M | 65 | 150 | 45.33 | 3.9 |
BMSC 07 | F | 75 | 150 | 23.66 | 4.3 |
BMSC 08 | F | 75 | 150 | 20.33 | 2.5 |
BMSC 09 | F | 64 | 280 | 45 | 4.8 |
BMSC 10 | M | 71 | 250 | 48 | 1.8 |
BMSC 11 | M | 57 | 370 | 31 | 3.8 |
BMSC 12 | F | 54 | 210 | 20.33 | 3.3 |
BMSC 13 | F | 74 | 540 | 23 | 4.0 |
BMSC 14 | F | 70 | 250 | 31.33 | 3.3 |
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TNC: total nucleated cells; CFU-F: colony-forming unit-fibroblast.
2.3. Preparation of Total Bone Marrow Cell Suspensions
Acetabular bone fragments were processed immediately after harvesting or after a maximum of 12 h of storage at 2-8°C in α-minimum essential medium (α-MEM, LGC Biotechnology, São Paulo, SP, BRA) supplemented with 20% fetal bovine serum (FBS, Gibco-Thermo Fisher, Waltham, MA, USA) [37, 48–50]. The samples were added to 50 mL tubes, and phosphate-buffered saline (PBS, Amresco, Solon, OH, USA) was added at a 1 : 4 (
2.4. Colony-Forming Efficiency (CFE) Assay
To estimate the number of colony-forming units (CFU-Fs) in the obtained marrow suspensions, nucleated cells were plated in triplicate at a density of
2.5. BMSC Isolation
To determine the optimal cell seeding density for BMSC isolation, nucleated cells from samples 01 to 04 were resuspended at
2.6. Large-Scale BMSC Expansion
Large-scale BMSC expansion was performed in multilayer cell factories, in a two-step process [37, 45]. In the first expansion cycle (passage 1—P1), BMSCs were seeded in 2-layer cell factories (1264 cm2, Corning Incorporated) at a density of
For the second expansion cycle, BMSCs were seeded in 10-layer cell factories (6320 cm2, Corning Incorporated) at a density of
2.7. Population Doubling Analysis
In each expansion cycle, the number of population doublings (PD) was calculated using the formula
2.8. Cryopreservation and Storage
A total of
2.9. Viability of the Cryopreserved Cells
After four and 40 weeks of cryopreservation in the aforementioned conditions, one vial from five BMSC products (07–11) was thawed in a water bath at 37°C. The cell suspension was immediately diluted 1 : 10 (
2.10. Bacterial, Endotoxin, and Mycoplasma Contamination Testing
To evaluate the sterility of the final BMSC products, the presence of aerobic and anaerobic bacteria and mycoplasma and the levels of endotoxin were evaluated. For aerobic and anaerobic bacterial contamination testing, 1 mL of the cell culture media collected at the end of bone marrow cell suspension preparations and at the end of P0 (isolation) and P2 was inoculated into BD BACTEC™ Plus Aerobic/F and BD BACTEC™ Plus Anaerobic/F (Becton Dickenson, New Jersey, USA) culture vials and incubated for 10 days.
Mycoplasma contamination and endotoxin levels were evaluated in P2 culture media with the detection kits MycoAlert™ and LAL Pyrogent™-5000 (both from Lonza, Basel, SWI), respectively. Measurements were performed in accordance with the manufacturer’s protocol.
2.11. Quantification of Bovine Transferrin
Levels of FBS proteins in the final cell products were estimated by the quantification of bovine transferrin concentration by ELISA. After the cells’ washing step with Ringer’s lactate, a 2 mL sample of the cell suspension was collected and measurements were performed according to the manufacturer’s instructions (Abnova, Taipei, TWN).
2.12. Immunophenotyping
For immunophenotypic characterization of cells,
2.13. In Vitro Osteogenic Differentiation and Von Kossa Staining
BMSCs were plated at a density of
2.14. In Vitro Adipogenic Differentiation and Oil Red O Staining
BMSCs were plated as described above. After reaching 100% confluence, the cells were incubated with α-MEM containing 0.5 mM isobutylmethylxanthine, 200 mM indomethacin, 10-6 M dexamethasone (all from Sigma), and 10 mM insulin (Humulin®, Lilly, São Paulo, SP, BRA) supplemented with 20% FBS for 21 days, and the medium was changed every 3 days [51]. To confirm the lipidic composition of the cell vacuoles, BMSCs were fixed in 4% paraformaldehyde for 10 minutes at room temperature, washed with propylene glycol PA (Vetec Quimica, Rio de Janeiro, RJ, BRA), and incubated with 0.5% Oil Red O solution (Sigma) in propylene glycol for 20 minutes. After two washes with 85% propylene glycol, the cells were photographed using a Nikon Eclipse TS100 inverted microscope.
2.15. In Vitro Chondrogenic Differentiation
BMSCs were resuspended in α-MEM supplemented with 20% FBS at a density of
2.16. Type II Collagen Immunofluorescence
To confirm type II collagen deposition in the chondrogenically induced micromasses, 5 μm paraffin sections were obtained as described above and incubated in citrate buffer (pH 6.0) at 96°C for 40 minutes. After blocking with 10% BSA for 1 h, the sections were incubated overnight at 4°C with an anti-collagen type II primary antibody (sc-288887, Santa Cruz Biotechnology) diluted 1 : 50 in Tris-buffered saline (TBS) with 1% BSA. Subsequently, the slides were washed three times with TBS and incubated for 2 h with an Alexa Fluor 546-conjugated secondary antibody (Life Technologies, Thermo Fisher) in the dark at room temperature. The nuclei were stained with 1 μg/mL DAPI solution (sc-3598, Santa Cruz Biotechnology). Fluorescence images were obtained using a Leica TCS SP5 laser scanning confocal microscope (Leica Microsystems, Wetzlar, GER).
2.17. Subcutaneous Xenotransplantation Assay
To evaluate the in vivo bone-forming potential of the expanded BMSCs,
2.18. Implant Histology and Immunohistochemistry
Following harvesting, the implants were fixed for 24 h in 4% paraformaldehyde in PBS. For routine histology, the implants were decalcified by incubation in 10% nitric acid (Vetec) for 3 days, processed for paraffin embedding, cut into 6 μm sections, and stained with H & E.
For immunohistochemistry, the implants were decalcified in 10% EDTA (Sigma) for 8-12 weeks and processed for paraffin embedding and sectioning as previously described. The sections were incubated overnight at 4°C with rabbit anti-lamin A/C antibody (M00438, Boster, Pleasanton, CA, USA) diluted 1 : 100 or with mouse anti-collagen type I antibody (Abcam, Cambridge, UK) diluted 1 : 300. After 2 washes with EnVision™ FLEX Wash Buffer (DAKO Agilent, Santa Clara, CA, USA), the sections were incubated for 2 h with EnVision Flex (DAKO Agilent). The signal was developed in EnVision Flex Substrate Buffer containing 20 μL/mL DAB—EnVision Flex DAB+ Chromogen (DAKO Agilent)—for 3 min. All images of the glass slides were obtained by digital scanning with an Aperio CS2 scanner and ImageScope software (both from Leica Biosystems).
2.19. Assessment of Bone Density by Micro-CT
Micro-CT scans were performed using the desktop SkyScan1172 scanner (Bruker, Brussels, BEL). One implant from each sample was placed in customized tubes and wrapped in gauze dampened with 4% formalin to avoid shrinkage. The scanning parameters were as follows: 4.0 μm isotropic pixel size and an X-ray source with 70 kV accelerating voltage and 141 μA current with a 500 μm Al filter. The samples were rotated 180° around their vertical axis with a rotational step of 0.4°, an exposure time of 1770 ms, and frame averaging of 3. The total scan time per sample was approximately 1.2 h. The images were reconstructed with NRecon v.1.7.1.0 software (Bruker) using a filtered back-projection algorithm. The 3D images were rendered using CTVox software (Bruker). To discriminate bone from HA/TCP, the grayscale images were binarized. For binarization, the threshold value was chosen based on a visual comparison of the original grayscale image and the binary image for each tissue slice. The binary samples were quantified in CTAn software (Bruker).
2.20. Statistical Analysis
The values were expressed either individually and/or as the mean/median, as appropriate. The Gaussian distribution was evaluated by the Shapiro-Wilk and Kolmogorov-Smirnov tests. Comparisons between groups were evaluated either by one-way ANOVA with Tukey’s multiple comparisons test or by Kruskal Wallis with Dunn’s multiple comparisons test.
3. Results
3.1. Patient and Sample Characteristics
Trabecular bone containing marrow was collected from nine females and five males with ages ranging between 48 and 75 years old (Table 1). The frequency of clonogenic cells in the samples was estimated by the colony-forming efficiency (CFE) assay. The average number of colonies harvested from each sample was heterogeneous and ranged from 7.66 to 48.0 CFU-Fs per 100000 nucleated cells (mean of
3.2. Optimization of Cell Seeding Density for BMSC Isolation
To determine the optimal conditions for improved BMSC isolation from the bone marrow total nucleated cell fractions, BMSC samples 01–04 were seeded at three cell densities:
[figures omitted; refer to PDF]
3.3. BMSC Manufacturing
BMSC manufacturing was performed in three steps: isolation in T-75 flasks (P0), followed by two rounds of expansion in 2- and 10-layer cell factories (Figure 2). The average BMSC harvest per T-75 flask at the end of P0 was
[figure omitted; refer to PDF]
Table 2
Yield (number) and viability of BMSCs at each culture step.
BMSC isolation (P0) | Passage 1 (P1) | Passage 2 (P2) | ||||||||||
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Cells seeded (106)/T-75 | Yield (106)/T-75 | %viability | Cells seeded (106) | Yield (106) | %viability | Time (days) | Cells seeded (106) | Yield (106) | %viability | Time (days) | Total production time (days) | |
BMSC 05 | 3.0 | 0.35 | 100 | 2.5 | 42.0 | 95 | 6 | 12 | 118.0 | 95 | 7 | 27 |
BMSC 06 | 3.0 | 0.92 | 90.5 | 2.5 | 30.0 | 98.1 | 3 | 12 | 56.0 | 95.2 | 6 | 23 |
BMSC 07 | 3.0 | 0.72 | 100 | 2.5 | 43.7 | 100 | 3 | 12 | 34.0 | 100 | 6 | 23 |
BMSC 08 | 3.0 | 0.52 | 92.3 | 2.5 | 12.0 | 96 | 4 | 12 | 40.0 | 98.7 | 5 | 23 |
BMSC 09 | 3.0 | 0.62 | 87.9 | 2.5 | 27.0 | 92.8 | 7 | 12 | 400.0 | 85.6 | 7 | 28 |
BMSC 10 | 3.0 | 0.37 | 100 | 2.5 | 12.0 | 90 | 7 | 12 | 26.0 | 92 | 7 | 28 |
BMSC 11 | 3.0 | 0.32 | 96.7 | 2.5 | 12.1 | 90.2 | 7 | 12 | 60.0 | 89.5 | 7 | 28 |
BMSC 12 | 3.0 | 0.41 | 97.3 | 2.5 | 15.0 | 98 | 7 | 12 | 20.0 | 97.5 | 7 | 28 |
BMSC 13 | 3.0 | 0.37 | 93.7 | 2.5 | 12.5 | 98.7 | 7 | 12 | 40.0 | 96.1 | 7 | 28 |
BMSC 14 | 3.0 | 0.31 | 98.3 | 2.5 | 13.0 | 94.1 | 7 | 12 | 36.5 | 85.7 | 7 | 28 |
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In P1, the cells reached 70% confluence in
3.4. Analysis of Population Doubling
With the adopted procedures for BMSC manufacturing, cells doubled an average of
Table 3
Number of population doublings and the doubling time for each BMSC passage.
Population doubling | Time for population doubling (days) | |||||||
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P0 | P1 | P2 | Cpd | P0 | P1 | P2 | ||
BMSC 05 | 9.75 | 4.07 | 3.30 | 17.12 | 1.43 | 1.47 | 2.12 | |
BMSC 06 | 3.23 | 3.58 | 2.22 | 9.04 | 4.33 | 0.84 | 2.70 | |
BMSC 07 | 12.31 | 4.13 | 1.50 | 17.94 | 1.13 | 0.73 | 3.99 | |
BMSC 08 | 12.05 | 2.26 | 2.00 | 16.31 | 1.16 | 1.77 | 2.50 | |
BMSC 09 | 11.85 | 3.43 | 5.06 | 20.34 | 1.18 | 2.04 | 1.38 | |
BMSC 10 | 11.02 | 2.26 | 1.50 | 15.51 | 1.26 | 3.09 | 4.66 | |
BMSC 11 | 11.74 | 2.28 | 2.58 | 16.60 | 1.19 | 3.08 | 2.71 | |
BMSC 12 | 11.99 | 2.58 | 0.74 | 15.31 | 1.16 | 2.71 | 9.50 | |
BMSC 13 | 12.08 | 2.32 | 2.00 | 16.04 | 1.15 | 3.01 | 3.50 | |
BMSC 14 | 11.37 | 2.38 | 1.99 | 15.74 | 1.23 | 2.94 | 3.52 | |
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Median | 1.18 | 2.37 |
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Cpd: cumulative population doubling.
3.5. Sterility Analysis of BMSC Products
To attest the sterility of bone samples and BMSCs and ultimately the quality of the technical procedures and the whole facility environment, tests for aerobic and anaerobic pathogens were performed at P0 and P2 in cell supernatants. No bacterial growth was ever detected (Table 4). The tests for mycoplasma and pyrogenic substances were also all negative (Table 4).
Table 4
Purity and safety analysis of the BMSCs.
Anaerobic and aerobic bacteria |
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BMSC 05 | ND | 0.57 | <0.005 | 7.43 |
BMSC 06 | ND | 0.43 | <0.005 | 0.04 |
BMSC 07 | ND | 0.33 | <0.005 | 0.04 |
BMSC 08 | ND | 0.42 | <0.005 | 0.03 |
BMSC 09 | ND | 1.08 | <0.005 | 0.04 |
BMSC 10 | ND | 0.82 | <0.005 | 0.05 |
BMSC 11 | ND | 0.37 | <0.005 | ND |
BMSC 12 | ND | 0.47 | <0.005 | ND |
BMSC 13 | ND | 0.63 | <0.022 | ND |
BMSC 14 | ND | 0.70 | <0.005 | ND |
ND: not detected.
3.6. Assessment of Animal Protein Content in Final BMSC Products
At the end of expansion, BMSCs were washed five times with Ringer’s lactate solution containing 0.5% human albumin. Then, the levels of residual animal-derived proteins in the final cell products were evaluated by the quantification of bovine transferrin. None of the samples showed a level above the limiting value of 10 ng/mL (Table 4), indicating that the BMSC products were compliant with GMP conditions.
3.7. Immunophenotypic Characterization and In Vitro Differentiation of Expanded BMSCs
To characterize the cell products, we first assessed the expression of BMSC-related cell surface markers by FACS. The expanded BMSCs homogenously expressed CD73, CD90, CD105, and CD146 and were negative for the hematopoietic lineage markers CD34, CD45, CD14, and CD11b (Table 5 and Supplementary Figure 1). Next, we evaluated their differentiation potential in vitro. The induced BMSCs were able to differentiate into osteoblasts, adipocytes, and chondrocytes (Figure 3 and Supplementary Figure 2), as shown by the deposition of mineralized nodules with positive Von Kossa staining (Figure 3(b)), intracellular lipid accumulation with positive Oil Red O staining (Figure 3(c)), and collagen type II-rich cartilaginous matrix as revealed by Masson’s Trichrome stain (Figures 3(e) and 3(f)) and immunofluorescence analysis (Figures 3(g)–3(i)).
Table 5
Immunophenotypic characterization of the lots of BMSCs that were produced.
%CD73 | %CD90 | %CD105 | %CD146 | %CD73/CD90/CD105/CD146 | %CD11b | %CD14 | %CD34 | %CD45 | |
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BMSC 05 | 99.8 | 99.8 | 62.3 | 99.7 | 70.2 | 0.59 | 2.20 | 1.34 | 3.21 |
BMSC 06 | 100 | 100 | 82.3 | 99.9 | 82.3 | 0.86 | 1.85 | 0.56 | 2.85 |
BMSC 07 | 100 | 100 | 83.3 | 99.1 | 73.4 | 2.38 | 0.69 | 0.16 | 3.32 |
BMSC 08 | 97.6 | 97.6 | 100 | 100 | 95 | 0.25 | 0.11 | 0.45 | 0.98 |
BMSC 09 | 100 | 98.1 | 66.4 | 98.3 | 50.1 | 0.69 | 1.24 | 0.55 | 2.39 |
BMSC 10 | 92.7 | 99.6 | 99.4 | 99.3 | 98.8 | 0.62 | 0.18 | 0.22 | 2.85 |
BMSC 11 | 99.7 | 97.1 | 95.8 | 88.4 | 62.9 | 1.35 | 1.14 | 2.17 | 2.67 |
BMSC 12 | 99.9 | 99.4 | 75.2 | 100 | 86.1 | 1.03 | 0.11 | 0.11 | 1.26 |
BMSC 13 | 100 | 100 | 89.2 | 100 | 72.2 | 3.56 | 0.19 | 0.80 | 4.18 |
BMSC 14 | 99.7 | 98.9 | 98.3 | 99.1 | 82.9 | 0.12 | 0.08 | 1.09 | 0.26 |
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[figures omitted; refer to PDF]
3.8. Assessment of the In Vivo Bone-Forming Potential
To verify whether the BMSCs in the final cell products indeed retained the ability to differentiate and form bone in vivo, the cells were subcutaneously transplanted into immunodeficient mice. The histological examination of the implants revealed the formation of ossicles for nine out of 10 BMSC products (Table 6).
Table 6
Osteogenic potential of the lots of BMSCs produced.
Tissues formed | Bone density (HU) |
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BMSC 05 | Bone/bone marrow | 1572 |
BMSC 06 | Bone | 1233 |
BMSC 07 | Bone/bone marrow | 994 |
BMSC 08 | Bone | 1209 |
BMSC 09 | Bone | 1605 |
BMSC 10 | Bone/bone marrow | 1363 |
BMSC 11 | Bone | 1946 |
BMSC 12 | Bone | 1074 |
BMSC 13 | Bone/bone marrow | 1356 |
BMSC 14 | Fibrous tissue | — |
The bone matrix was deposited over the surfaces of the HA/TCP particles and resembled trabecular bone architecture (Figures 4(b), 4(d), and 4(f)). Osteocytes were embedded in the newly synthesized matrix (Figure 4(b), arrowheads), and the reconstituted marrow stroma was filled with hematopoietic cells (Figure 4(b), asterisk). The human origin of the cells inside the ossicles was corroborated through human lamin A/C and type I collagen detection within the woven bone (Figures 4(d) and 4(f) and Supplementary Figure 3). Micro-CT-based 3D reconstruction of the BMSC implants (Supplementary Figure 4) showed that the neoformed bone density ranged from 994 to 1946 HU (Hounsfield unit), which was in accordance with the reference density values described for human cancellous and cortical bone (Table 6).
[figures omitted; refer to PDF]
3.9. Analysis of Cryopreservation Conditions
Finally, to evaluate the quality of the cryopreservation procedures, vials of five BMSC lots were thawed after four and 40 weeks of storage. Cell viability was similar at both time points and did not significantly differ from the percent viability at the time of cryopreservation (Table 7).
Table 7
Viability evaluation of the cryopreserved lots of BMSCs.
At the time of cryopreservation (%) | After 4 weeks of cryopreservation (%) | After 40 weeks of cryopreservation (%) | |
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BMSC 07 | 100 | 78 | 85 |
BMSC 08 | 98.7 | 92 | 84 |
BMSC 09 | 85.6 | 93 | 95 |
BMSC 10 | 92 | 91 | 75 |
BMSC 11 | 89.5 | 88 | 78 |
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4. Discussion
Because of their osteogenic, immunomodulatory, and angiogenesis-promoting potential, BMSCs have been the focus of extensive research of their clinical application in orthopedics [59]. However, the translation to the bedside still faces important bottlenecks, one being the lack of regulatory and technical consensus determining the overall conditions that should be adopted for BMSC manufacturing and what assays should be performed in order to validate the cell potency [25, 26]. In this sense, centers in different countries have adopted its own procedures, resulting in the manufacturing of BMSC products with distinct gene expression signatures and functional potentials. In Brazil, the sanitary regulatory health agency (ANVISA) provided general regulatory rules for the establishment of facilities for cell manufacturing and/or processing for therapeutic purposes but left to the facilities the decision about what technical procedures should be adopted for primary tissue harvesting, processing, cell isolation, expansion, and characterization, according to the particularities of the cells to be manufactured. In this report, we described our procedures to generate functionally validated BMSC products that conform to both GMP standards and national regulatory policies.
In order to have a parameter for comparison, our protocols were based on the previous experience of the NIH BMSC bank [37]. But differently from their protocol, in which cells were expanded in three cycles (one in 2-layer and two in 10-layer cell factories), in our manufacturing process, cells were expanded in only two subsequent rounds in 2- and 10-layer cell factories, respectively. Another difference was the source of bone marrow. Instead of using bone marrow aspirates from volunteered donors, we used trabecular bone discards. The main reason for this choice was the fact that at this initial point our goal was simply to set and evaluate the procedures for BMSC manufacturing in our in-house facility. Because we are housed in an orthopedic hospital, bone discards from primary total hip arthroplasties (THA) are always available and can be obtained without subjecting donors to any additional procedures. And from previous studies of our group using BMSCs isolated from THA as a model, the capacity of these cells to proliferate and differentiate in vitro and in vivo was already known [50], thus proving its usefulness to validate our procedures.
Indeed, the number of clonogenic cells in the bone marrow of the THA samples, as assessed by the CFE assay, was within the previously reported range of CFE measured in bone specimens from eight healthy adults [48] and was, on average, three times higher than the CFE of bone marrow aspirates reported in the NIH BMSC bank manuscript [37]. This was not surprising because marrow aspirates are usually more contaminated with peripheral blood, which dilutes the marrow, decreasing CFE counts. Also, BMSCs were successfully isolated from all 14 samples.
Although the time for population doubling progressively increased during expansion—which can be a reflex of the donors’ age—the total average cell yield obtained using just one cell factory per cycle (our minimal infrastructure capacity) showed that a significant number of cells can be produced with our protocol, considering a lot unit of
Because ANVISA and international regulatory organizations, such as the US Food and Drug Administration (FDA), have recommended that reagents from animal sources should be avoided when manufacturing cells for human use because of the risk of zoonoses and xenogeneic immunological reactions, efforts have been made in the field to identify substitutes for fetal bovine serum, such as chemically defined media [60–63], human serum [64], and activated platelet lysates [65–68]. Although platelet lysates were shown to induce BMSC proliferation as efficiently as FBS [43, 66], strong evidence that these BMSC products have similar functional potency as FBS-expanded BMSCs has not been provided so far. Indeed, Ren and colleagues [65] showed that the substitution of FBS with platelet lysates during BMSC isolation and expansion resulted in BMSCs with different genes and microRNA expression profiles. While FBS-cultured BMSCs expressed genes involved in the MAPK, TGF-β, adhesion, and extracellular matrix pathways, BMSCs cultured with platelet lysates expressed genes related to metabolic, proliferation, cell cycle, and immune response pathways. Functional assays with either of these BMSC products were not performed [65]. Therefore, in line with the standard protocol used by the NIH cell bank [37], we included FBS in the BMSC isolation and expansion media but added washing steps for the final cell products to reduce the animal protein content. The BMSC lots were shown to have animal protein concentrations below the threshold limit stipulated by the FDA, indicating that the cell products had no increased risks of infection that would hamper their use in patients.
Finally, because at present no known phenotypic or genetic characteristic allows the prospective determination of the differentiation ability of a given population of BMSCs once transplanted in vivo [69, 70] and in vitro assays are believed to have a high probability of being artifactual [49, 71–73], we established the in vivo differentiation assay as the critical test to ascertain BMSC potency. Although the in vivo assay takes months to complete and to obtain a result, it is long recognized by scientists of the field as the best available method to evaluate the intrinsic differentiation potential of a given cell population, as it allows the inherent expression of cell potency without any exogenous chemical inducer [21, 49, 58, 74, 75]. By using this functional quality control method, we showed that nine out of 10 of our BMSC products formed bone in vivo with the expected histology and matrix microarchitecture. Additionally, of the nine samples that formed bone, four developed a supportive hematopoietic stroma, which is an indicator of the presence of multipotential skeletal stem cells in a BMSC population [20, 49, 70, 76]. Therefore, we concluded that the protocol we established for large-scale BMSC manufacturing preserved the potential of cells to form bone in vivo, and we confirmed its suitability for the future generation of cells for bone repair strategies. This protocol can be equally used for BMSC isolation from any source sample. The only adaptation needed if the source sample is a bone marrow aspirate is the adjustment of the initial total nucleated cell seeding density for BMSC isolation, which needs to be higher because of the decreased CFE counts of the aspirates. For this adaptation, we refer the reader to the report of the NIH BMSC bank [37].
Although we have shown that BMSCs isolated from THA bone discards can form bone in vivo, which suggests that these samples could be useful for BMSC banking for allogeneic use, we emphasize that the choice for autologous or allogeneic cell applications, as well as the decision of the best bone marrow source material, needs to be carefully evaluated on a case by case basis. Because cell application itself was beyond our present objective, we did not conduct immunogenicity and/or HLA compatibility tests, but in any allogenic strategy scenario, these tests should be conducted, in addition to the tests presented herein.
5. Conclusion
Due to the implementation of regulatory rules for the establishment of facilities for cell manufacturing by the Brazilian sanitary agency, a significant step towards the clinical use of BMSCs in orthopedics was taken. However, definition of the procedures for the large-scale production of clinical-grade BMSCs with validated bone-forming potential in vivo is still needed. In this study, we described a protocol for the generation of BMSC products with certified phenotype and capacity to form bone after in vivo transplantation, which will serve as the basis for in-house BMSC manufacturing for future clinical applications in our center.
Acknowledgments
This study was funded by the National Institute of Traumatology and Orthopedics and the Brazilian Ministry of Health.
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Abstract
In vitro-expanded bone marrow stromal cells (BMSCs) have long been proposed for the treatment of complex bone-related injuries because of their inherent potential to differentiate into multiple skeletal cell types, modulate inflammatory responses, and support angiogenesis. Although a wide variety of methods have been used to expand BMSCs on a large scale by using good manufacturing practice (GMP), little attention has been paid to whether the expansion procedures indeed allow the maintenance of critical cell characteristics and potency, which are crucial for therapeutic effectiveness. Here, we described standard procedures adopted in our facility for the manufacture of clinical-grade BMSC products with a preserved capacity to generate bone in vivo in compliance with the Brazilian regulatory guidelines for cells intended for use in humans. Bone marrow samples were obtained from trabecular bone. After cell isolation in standard monolayer flasks, BMSC expansion was subsequently performed in two cycles, in 2- and 10-layer cell factories, respectively. The average cell yield per cell factory at passage 1 was of
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1 Master Program in Musculoskeletal Sciences, National Institute of Traumatology and Orthopedics, Rio de Janeiro 20940-070, Brazil; Research Division, National Institute of Traumatology and Orthopedics, Rio de Janeiro 20940-070, Brazil
2 Research Division, National Institute of Traumatology and Orthopedics, Rio de Janeiro 20940-070, Brazil; Trauma Center, National Institute of Traumatology and Orthopedics, Rio de Janeiro 20940-070, Brazil
3 Hip Surgery Center, National Institute of Traumatology and Orthopedics, Rio de Janeiro 20940-070, Brazil
4 Institute of Paediatrics and Puericulture Martagão Gesteira, Federal University of Rio de Janeiro, Rio de Janeiro 21941-912, Brazil
5 Institute of Clinical Dentistry, University of Oslo, Oslo 0317, Norway
6 Institute of Biophysics Carlos Chagas Filho, Federal University of Rio de Janeiro, Rio de Janeiro 21941-902, Brazil; Texas Heart Institute, Regenerative Medicine Research, Texas 77030, USA
7 Research Division, National Institute of Traumatology and Orthopedics, Rio de Janeiro 20940-070, Brazil
8 Graduate Program in Pathology, Fluminense Federal University, Rio de Janeiro 24030-215, Brazil
9 Department of Radiology, Clementino Fraga Filho University Hospital, Federal University of Rio de Janeiro, Rio de Janeiro 21941-913, Brazil