Tissue Engineering Part C: Methods One-step purification of human skeletal muscle myoblasts and subsequent expansion using laminin-coated surface (doi: 10.1089/ten.TEC.2015.0015) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
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1 One-step purification of human skeletal muscle myoblasts and subsequent expansion using laminin-coated surface
Shiplu Roy Chowdhury, Ph.D.,1* Annis binti Ismail, M.D.,1 Sia Chye Chee, M.D.,1 Mohd Suffian bin Laupa, M.D.,1 Fadhlun binti Jaffri, M.D.,1 Salfarina Ezrina Mohmad Saberi, M.S.,1 and Ruszymah Bt Hj Idrus, Ph.D.1,2
1
Tissue Engineering Centre, Faculty of Medicine, Universiti Kebangsaan Malaysia Medical
Centre, Universiti Kebangsaan Malaysia, 56000 Kuala Lumpur, Malaysia 2
Department of physiology, Faculty of Medicine, Universiti Kebangsaan Malaysia, 50300 Kuala
Lumpur, Malaysia
Corresponding author: Dr. Shiplu Roy Chowdhury Tissue Engineering Center, Universiti Kabangsaan Malaysia Medical Center Jalan Yaacob Latif, Bandar Tun Razak, Cheras 56000, Kuala Lumpur, Malaysia Tel: +603 9145 7679; Fax: +603 9145 7678 E-mail: [email protected]
1
Tissue Engineering Part C: Methods One-step purification of human skeletal muscle myoblasts and subsequent expansion using laminin-coated surface (doi: 10.1089/ten.TEC.2015.0015) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
Page 2 of 29
2 ABSTRACT Skeletal myoblasts have been extensively used to study muscle growth and differentiation and were recently tested for their application as cell therapy and as gene delivery system to treat muscle and non-muscle diseases. However, contamination of fibroblasts in isolated cells from skeletal muscle is one of the long-standing problems for routine expansion. This study aimed to establish a simple one-step process to purify myoblasts, and maintain their purity during expansion. Mixed cells were preplated serially on laminin- and collagen type I-coated surfaces in a different array for 5, 10 and 15 minutes. Immunocytochemical staining with antibodies specific to myoblasts was performed to evaluate myoblast attachment efficiency, purity and yield. It was found that laminin-coated surface favors the attachment of myoblasts. The highest myoblast purity of (78.9±6.8)% was achieved by 5 minutes of preplating only on the laminin-coated surface with a yield of (56.9±3.3)%. Primary cells, isolated from skeletal muscle (n=4), confirms the enhancement of purity via preplating on laminin-coated surface for 5 minutes. Subsequent expansion after preplating enhanced myoblast purity due to an increase in myoblast growth than fibroblasts. Myoblast purity was achieved approximately 98% when another preplating was performed during passaging. In conclusion, myoblasts can be purified and efficiently expanded in one step by preplating on laminin-coated surface, which is a simple and robust technique.
Keywords: myoblasts purification, preplating, bioprocess design, myoblast expansion, tissue engineering
2
Tissue Engineering Part C: Methods One-step purification of human skeletal muscle myoblasts and subsequent expansion using laminin-coated surface (doi: 10.1089/ten.TEC.2015.0015) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
Page 3 of 29
3 1.
INTRODUCTION Since their discovery in 1961 by Mauro, skeletal myoblasts have been extensively used in
culture to study muscle growth and differentiation [1]. Over the past decades, myoblasts have gained remarkable attention for their clinical application due to their easy accessibility and availability, in vitro self-renewal capacity, stress resistance and lack of tumorigenicity [2, 3]. Myoblasts have been tested for their application as a cell therapy to regenerate muscles [2-9] and as a gene delivery system [10] to treat muscle and non-muscle diseases. Several preclinical and clinical studies have shown the functional benefits of myoblast transplantation in the treatment of congenital muscle disorder [4, 10], cardiac diseases [2, 3, 5, 6], urinary incontinence [7] and traumatic muscle damage [8, 9]. Despite multiple applications of myoblasts in both basic research and clinics, one of the long-standing challenges for the routine culture of myoblasts in vitro is the contamination of fibroblasts during the isolation of cells from skeletal muscle. This fibroblast population increases dramatically during expansion, as they proliferate faster than myoblasts. To avoid this problem, basic research on myoblasts has commonly used stable cell lines, although these cells lack typical myoblast morphological and functional properties [11]. However, for clinical application, primary myoblasts from autologous and allologous origin are used, and they need to be expanded in vitro to achieve adequate amounts of cells for transplantation. Fibroblast overgrowth during expansion significantly reduces myoblast purity and requires purification prior to clinical application. Several myoblast purification procedures have been reported in the literature, including serial preplating [12, 13], selective adhesion of myoblasts and fibroblasts [14], percoll density centrifugation [15],cell sorting by size [16] and antibody tagging [17, 18] and supplementation of 3
Tissue Engineering Part C: Methods One-step purification of human skeletal muscle myoblasts and subsequent expansion using laminin-coated surface (doi: 10.1089/ten.TEC.2015.0015) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
Page 4 of 29
4 fibroblast growth inhibitor [18, 19]. Cell sorting techniques have been shown to increase myoblast yield but with relatively poor myoblast purity. In addition, these procedures are complicated and require specialized equipment. The usage of growth inhibitor, such as mitomycin C and irradiation, has also been tested to remove fibroblasts and has shown effective purification of myoblasts. However, these techniques reduce the proliferative activity of myoblasts and are not suitable for expansion. In contrast, the preplating technique is simple and is most commonly used to purify myoblasts. Preplating techniques traditionally utilize collagen type I-coated surfaces, which enable fibroblasts to attach faster than myoblasts, and the myoblast population is obtained after multiple preplatings [12, 13, 18]. However, the yield of myoblasts is low in conventional preplating techniques, and these techniques fail to completely remove fibroblasts. However, these techniques are not viable for use in cell expansion either due to an incapability of myoblast proliferation or a failure to completely remove fibroblasts. The presence of a small fraction of fibroblasts after purification may significantly reduce myoblast purity during expansion due to fibroblast overgrowth. This study aimed to develop a simple and effective pre-plating technique that not only purifies myoblasts but also maintains their purity during expansion. For this purpose, laminin-coated surfaces, which are known to facilitate myoblast attachment and proliferation [20, 21], were used alone or in-combination with conventional collagen type-I coated surfaces. The yield and purity of myoblasts were evaluated using immunocytochemical staining with myoblast-specific markers to optimize the preplating technique. Purification of several primary cells isolated from human skeletal muscle tissue was also tested to evaluate the effectivity of preplating technique. Moreover, the optimized preplating technique was applied for serial passages to evaluate the maintenance of purity during expansion. 4
Tissue Engineering Part C: Methods One-step purification of human skeletal muscle myoblasts and subsequent expansion using laminin-coated surface (doi: 10.1089/ten.TEC.2015.0015) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
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5
2.
MATERIALS AND METHODS
2.1 Muscle cells harvesting and culture This research was approved by Universiti Kebangsaan Malaysia Research and Ethics Committee (UKMREC) with approval code of FF-037-2013 and FF-313-2010. Cryopreserved primary human skeletal muscle myoblast cells (HSMMs; Lonza, USA) and cells harvested from human skeletal muscle tissue were used in this study. HSMMs of passage 2 were thawed and seeded into a 75-cm2 tissue culture flask with F10:DMEM (1:1, Sigma, USA) containing 10% FBS (PAA Laboratories, Austria), prior to incubation. All of the cell incubations in this study were performed at 37°C with an atmospheric condition of 5% CO2. Waste medium was replaced every 48 hours with fresh culture medium. After reaching 80% confluence, the cells were treated with trypsin-EDTA (Mediatech, USA) for 5 minutes to detach the cells from the culture surface. The cell suspension was then centrifuged and resuspended in fresh culture medium and used for preplating experiments (at passage 4). Human skeletal muscle samples were collected as redundant tissue from four consented patients undergoing lower limb surgery. The samples were processed within 24 hours after surgery. Muscle tissues were clean from fat, connective tissue and blood vessel. Tissue samples were minced into small pieces and digested with 0.25% trypsin (Sigma) in 37°C incubator shaker for 10 minutes. Undigested tissue were separated by centrifugation at 500 rpm for 5 minutes. Supernatant were collected and neutralize with same volume of trypsin inhibitor. This digestion steps were performed for undigested tissue another 2-3 times. Finally, all the supernatant were centrifuge at 1000 rpm for 10 minutes. Pellet was resuspended with F10:DMEM containing 5
Tissue Engineering Part C: Methods One-step purification of human skeletal muscle myoblasts and subsequent expansion using laminin-coated surface (doi: 10.1089/ten.TEC.2015.0015) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
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6 10% FBS. Cells were seeded on 75 cm2 culture flask (Greiner, Germany) and incubate at 37°C with an atmospheric condition of 5% CO2. First medium change was performed 72-96 hours of seeding and subsequent changes were performed every 48 hours. After reaching 80% confluence, the cells were treated with trypsin-EDTA for 5 minutes. The cell suspension was then centrifuged and resuspended in fresh culture medium and used for preplating experiments (at passage 1). 2.2 Immune fluorescence (IF) staining Immune fluorescence staining was performed with anti-desmin and/or anti-fibroblast antibodies to identify myoblasts and fibroblasts, respectively. This technique was also used to evaluate myoblast and fibroblast attachment efficiency as well as myoblast purity and yield. Briefly, adherent cells were fixed with 4% paraformaldehyde (Sigma) and permeabilized with 0.05% triton X-100 (Sigma). After masking the nonspecific proteins with 10% goat serum (Gibco, USA), the cells were incubated for 1 h at room temperature with a mixture of rabbit antihuman desmin (1:250; Novus Biologicals, USA) and mouse anti-fibroblast clone TE-7 (1:100; Millipore, USA). Cells were then immunolabeled with a mixture of Alexa Fluor 594 goat antirabbit and Alexa Fluor 488 goat anti-mouse (1:250; Molecular Probes; USA), followed by counter staining with dye 4'-6-diamidino-2-phenylindole (DAPI; Molecular Probes). HSMMs and primary cells isolated from skeletal muscle tissue were also stained only for the anti-human desmin antibody using a similar protocol. At least five images were captured randomly from each plate using a Nikon A1 confocal microscope. Cells were manually quantified for total myoblasts (Desmin positive), total fibroblasts (fibroblast marker positive) and total cells (DAPI positive). In the case of single staining with Desmin, the total number of fibroblasts was calculated by subtracting the number of total myoblasts from the total number of cells. 6
Tissue Engineering Part C: Methods One-step purification of human skeletal muscle myoblasts and subsequent expansion using laminin-coated surface (doi: 10.1089/ten.TEC.2015.0015) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
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7 2.3 Surface coating with laminin and collagen type I Surface coating with laminin (Sigma) has been described elsewhere [21]. Briefly, 50 g/ml of laminin solution was added to the culture surface and incubated for 1-2 hours. Culture surfaces were washed with pre-warmed PBS (Sigma) prior to cell culture use. Rat tail collagen type I (Sigma) was used to coat the culture surface according to the manufacturer's protocol. Briefly, the culture surface was incubated with 250g/ml collagen type I for 2-3 hours. Excess fluid was then removed, and the culture flask was dried overnight at ambient temperature. The culture surface was washed with pre-warmed PBS prior to cell culture use. 2.4 Preplating Figure 1 demonstrates the preplating experiment on different orders of laminin- and collagen type I-coated surfaces. For the serial plating experiment, 2.5×103 cells/cm2 were seeded on the 1st plate (collagen type I- or laminin-coated surface) and incubated for 5, 10 or 15 minutes. Unattached cells were then transferred to the 2nd plate (collagen type I- or laminincoated surface) and similarly incubated either for 5, 10 or 15 minutes. Finally, the rest of the unattached cells were transferred onto the 3rd plate (plain surface). The attached cells after serial preplating on different surfaces were incubated for 24 hours prior to immunofluorescence (IF) staining. To evaluate the myoblast purity of cells that were used for preplating, the cells were seeded onto a plain surface without serial plating and incubated for 24 hours prior to IF staining (control). Preplating experiment with primary cells were performed only for the best condition to confirm the effectivity of the purification techniques. The purity and yield of myoblasts for each plate of preplating were evaluated using the following equations: 7
Tissue Engineering Part C: Methods One-step purification of human skeletal muscle myoblasts and subsequent expansion using laminin-coated surface (doi: 10.1089/ten.TEC.2015.0015) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
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8 Purity of myoblasts
Yield of myoblasts
No. of myoblasts on plate of interest 100% Total cells on the plate of interest
No. of myoblasts on plate of interest 100% Total no. of myoblasts on all 3 plates of preplating
2.5 Attachment efficiency Attachment efficiency of myoblasts and fibroblasts was evaluated on plain, collagen type Iand laminin-coated surface for the incubation period of 5, 10 or 15 minutes using preplating experiment as described in previous section. The conditions were listed in Fig 1. Efficiency of myoblast and fibroblast attachment was evaluated only at the 1st plate of serial pre-plating. Briefly, mixed population of myoblasts and fibroblasts were seeded on different culture surface (1st plate), and incubated for 5, 10 or 15 minutes to facilitate the attachment of myoblasts and fibroblasts. Unattached cells were transferred to subsequent plates as shown in Fig 1. Finally, adherent cells were incubated for another 24 hours, and IF staining was perform to evaluate number of attached myoblasts and fibroblasts (as describe in section 2.2). The efficiency of myoblast and fibroblast attachment for 5, 10 or 15 minutes incubation on different culture surface was evaluated via following equation: No. of myoblasts at 1st plate of preplating Total no. of myoblasts on 3 plates of preplating No. of fibroblast s at 1st plate of preplating Fibroblast attachment efficiency for incubation time t, Af, t Total no. of fibroblast s on 3 plates of preplating
Myoblast attachment efficiency for incubation time t, Am, t
2.6 Purification and subsequent cell expansion To evaluate the maintenance of myoblast purity during cell expansion, purification and expansion of myoblasts were performed on laminin-coated surface for 2 subsequent passages. Purification was performed via preplating of mixed cells on laminin-coated surface for 5 minutes, and attached cells on laminin-coated surface was expanded for 144 hours. Finally, cells 8
Tissue Engineering Part C: Methods One-step purification of human skeletal muscle myoblasts and subsequent expansion using laminin-coated surface (doi: 10.1089/ten.TEC.2015.0015) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
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9 were trypsinize and pre-plated onto laminin-coated surface for 5 minutes to perform another round of purification. Myoblast purity was evaluated at 0 hour (initial sample without purification), 24 hours (after 1st purification), 144 hours (after trypsinization without purification) and 168 hours (24 hours after 2nd purification) using protocol described in earlier sections. Moreover, growth rate of myoblasts and fibroblasts was also evaluated on laminin-coated surface along with that on plain surface. For this purpose, mixed population of cells were seeded on plain and laminin-coated surface at seeding density of 2.5×103 cells/cm2 and culture for 144 hours. Adherent cells at 24 and 144 hours were stained with anti-desmin and anti-fibroblasts (as described earlier) to determine the myoblasts and fibroblasts population, respectively. The growth rate of myoblasts and fibroblasts were evaluated using the following equationGrowth rate (h-1) = Ln (cell concentration at 144 h / cell concentration at 24 h) /120 h 2.8 Statistical Analysis All the experiment were performed in triplicate. The value was shown as mean ± standard deviation. Student’s t-tests were performed to determine statistical significance. P value less than 0.05 was considered as statistically significant.
3.
RESULTS
3.1 Identification of myoblasts and fibroblasts Myoblasts and fibroblasts in a mixed culture were identified via immunostaining as an invasive technique. Only two types of cells were identified in culture (Fig. 2). A group of cells 9
Tissue Engineering Part C: Methods One-step purification of human skeletal muscle myoblasts and subsequent expansion using laminin-coated surface (doi: 10.1089/ten.TEC.2015.0015) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
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10 was positive for only desmin and identified as myoblasts, whereas the other group was positive for only the fibroblast-specific marker and identified as fibroblasts. On the basis of this observation, for further experiments in this study, immunostaining was employed only with desmin, and desmin-positive cells were identified as myoblasts and desmin-negative cells were identified as fibroblasts. 3.2 Attachment efficiency of myoblasts and fibroblasts To evaluate the efficiency of myoblast and fibroblast attachment on plain, laminin- and collagen type I-coated surfaces in relationship to time, a mixed population of myoblasts and fibroblasts was seeded onto the designated culture surface and incubated for 5, 10 and 15 minutes. Representative images from each condition are shown in Figure 3A. It was found that myoblasts preferentially attached to the laminin-coated surface and that the efficiency of myoblast attachment (Am) on laminin-coated surfaces was significantly higher for an incubation period of 5, 10 and 15 minutes compared to fibroblasts (Fig. 3B). The attachment of myoblasts onto the laminin-coated surface was initiated immediately as the Am value for the 5-minute incubation was 0.58±0.06, which increased significantly to 0.81±0.02 for the 10-minute incubation. The value of Am for the 15-minute incubation showed a further increase (0.85±0.05) but was not significantly different from that of 10 minutes. It was also found that the efficiency of fibroblast attachment (Af) on laminin-coated surfaces increased significantly with an increase in incubation time and that the rate of the increase was comparatively higher compared to myoblasts. Fibroblast attachment on laminin-coated surface for 10 and 15 minutes incubation increased 2.8 and 3.6 folds compared to 5 minutes incubation, respectively, whereas for myoblasts increased 1.4 and 1.5 folds, respectively.
10
Tissue Engineering Part C: Methods One-step purification of human skeletal muscle myoblasts and subsequent expansion using laminin-coated surface (doi: 10.1089/ten.TEC.2015.0015) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
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11 In contrast to myoblasts, fibroblasts preferentially attached onto collagen type I-coated surfaces, and the Af on collagen type I-coated surfaces for 10- and 15-minute incubations was significantly higher compared to Am, although no significant differences were observed for the 5minute incubation (Fig. 3). The Af value on the collagen type I-coated surface increased sharply from 0.25±0.11 at 5 minutes of incubation to 0.78±0.08 at 10 minutes of incubation. No further increase was detected for the 15-minute incubation period. On a plain surface, the efficiency of myoblast and fibroblast attachment also increased with an increase in incubation time; however, no significant difference was observed for myoblast and fibroblast attachment. 3.3 Efficiency of myoblast purification To purify myoblasts, the serial plating technique was employed using HSMMs, as demonstrated schematically in Fig. 1. The quantitative evaluation for purity is demonstrated in Figure 4A. The highest purity of myoblasts was achieved by incubating the mixed population of cells on the laminin-coated surface for 5 minutes (1st plate of serial plating), and the purity was estimated to be ~79%, which was 1.7 times higher compared to the initial population and was statistically significant. However, the purity of myoblasts significantly decreased after 10- and 15-minute incubations on the laminin-coated surface (1st plate of serial plating). High purity myoblast populations were also obtained from two other conditions. In one case, two subsequent plating on collagen-coated surfaces for 10 minutes resulted in a myoblast purity of ~71% on a subsequent plate, i.e., on a plain surface. In another case, the cells incubated for 15 minutes first on the collagen type I-coated surface and then subsequently on the laminin-coated surface resulted in a purity of ~74% on the laminin-coated surface.
11
Tissue Engineering Part C: Methods One-step purification of human skeletal muscle myoblasts and subsequent expansion using laminin-coated surface (doi: 10.1089/ten.TEC.2015.0015) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
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12 To understand the effectiveness of the purification process, the yield of myoblasts in each condition was also evaluated (Fig. 4B). Yield of myoblasts on the laminin-coated surface (1st plate of serial plating) after the 5-minute incubation was ~57%. However, the yields of myoblasts on the laminin-coated surface with 10- and 15-minute incubations were 1.4 and 1.3 times higher compared to the 5-minute incubation, respectively (p
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1 One-step purification of human skeletal muscle myoblasts and subsequent expansion using laminin-coated surface
Shiplu Roy Chowdhury, Ph.D.,1* Annis binti Ismail, M.D.,1 Sia Chye Chee, M.D.,1 Mohd Suffian bin Laupa, M.D.,1 Fadhlun binti Jaffri, M.D.,1 Salfarina Ezrina Mohmad Saberi, M.S.,1 and Ruszymah Bt Hj Idrus, Ph.D.1,2
1
Tissue Engineering Centre, Faculty of Medicine, Universiti Kebangsaan Malaysia Medical
Centre, Universiti Kebangsaan Malaysia, 56000 Kuala Lumpur, Malaysia 2
Department of physiology, Faculty of Medicine, Universiti Kebangsaan Malaysia, 50300 Kuala
Lumpur, Malaysia
Corresponding author: Dr. Shiplu Roy Chowdhury Tissue Engineering Center, Universiti Kabangsaan Malaysia Medical Center Jalan Yaacob Latif, Bandar Tun Razak, Cheras 56000, Kuala Lumpur, Malaysia Tel: +603 9145 7679; Fax: +603 9145 7678 E-mail: [email protected]
1
Tissue Engineering Part C: Methods One-step purification of human skeletal muscle myoblasts and subsequent expansion using laminin-coated surface (doi: 10.1089/ten.TEC.2015.0015) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
Page 2 of 29
2 ABSTRACT Skeletal myoblasts have been extensively used to study muscle growth and differentiation and were recently tested for their application as cell therapy and as gene delivery system to treat muscle and non-muscle diseases. However, contamination of fibroblasts in isolated cells from skeletal muscle is one of the long-standing problems for routine expansion. This study aimed to establish a simple one-step process to purify myoblasts, and maintain their purity during expansion. Mixed cells were preplated serially on laminin- and collagen type I-coated surfaces in a different array for 5, 10 and 15 minutes. Immunocytochemical staining with antibodies specific to myoblasts was performed to evaluate myoblast attachment efficiency, purity and yield. It was found that laminin-coated surface favors the attachment of myoblasts. The highest myoblast purity of (78.9±6.8)% was achieved by 5 minutes of preplating only on the laminin-coated surface with a yield of (56.9±3.3)%. Primary cells, isolated from skeletal muscle (n=4), confirms the enhancement of purity via preplating on laminin-coated surface for 5 minutes. Subsequent expansion after preplating enhanced myoblast purity due to an increase in myoblast growth than fibroblasts. Myoblast purity was achieved approximately 98% when another preplating was performed during passaging. In conclusion, myoblasts can be purified and efficiently expanded in one step by preplating on laminin-coated surface, which is a simple and robust technique.
Keywords: myoblasts purification, preplating, bioprocess design, myoblast expansion, tissue engineering
2
Tissue Engineering Part C: Methods One-step purification of human skeletal muscle myoblasts and subsequent expansion using laminin-coated surface (doi: 10.1089/ten.TEC.2015.0015) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
Page 3 of 29
3 1.
INTRODUCTION Since their discovery in 1961 by Mauro, skeletal myoblasts have been extensively used in
culture to study muscle growth and differentiation [1]. Over the past decades, myoblasts have gained remarkable attention for their clinical application due to their easy accessibility and availability, in vitro self-renewal capacity, stress resistance and lack of tumorigenicity [2, 3]. Myoblasts have been tested for their application as a cell therapy to regenerate muscles [2-9] and as a gene delivery system [10] to treat muscle and non-muscle diseases. Several preclinical and clinical studies have shown the functional benefits of myoblast transplantation in the treatment of congenital muscle disorder [4, 10], cardiac diseases [2, 3, 5, 6], urinary incontinence [7] and traumatic muscle damage [8, 9]. Despite multiple applications of myoblasts in both basic research and clinics, one of the long-standing challenges for the routine culture of myoblasts in vitro is the contamination of fibroblasts during the isolation of cells from skeletal muscle. This fibroblast population increases dramatically during expansion, as they proliferate faster than myoblasts. To avoid this problem, basic research on myoblasts has commonly used stable cell lines, although these cells lack typical myoblast morphological and functional properties [11]. However, for clinical application, primary myoblasts from autologous and allologous origin are used, and they need to be expanded in vitro to achieve adequate amounts of cells for transplantation. Fibroblast overgrowth during expansion significantly reduces myoblast purity and requires purification prior to clinical application. Several myoblast purification procedures have been reported in the literature, including serial preplating [12, 13], selective adhesion of myoblasts and fibroblasts [14], percoll density centrifugation [15],cell sorting by size [16] and antibody tagging [17, 18] and supplementation of 3
Tissue Engineering Part C: Methods One-step purification of human skeletal muscle myoblasts and subsequent expansion using laminin-coated surface (doi: 10.1089/ten.TEC.2015.0015) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
Page 4 of 29
4 fibroblast growth inhibitor [18, 19]. Cell sorting techniques have been shown to increase myoblast yield but with relatively poor myoblast purity. In addition, these procedures are complicated and require specialized equipment. The usage of growth inhibitor, such as mitomycin C and irradiation, has also been tested to remove fibroblasts and has shown effective purification of myoblasts. However, these techniques reduce the proliferative activity of myoblasts and are not suitable for expansion. In contrast, the preplating technique is simple and is most commonly used to purify myoblasts. Preplating techniques traditionally utilize collagen type I-coated surfaces, which enable fibroblasts to attach faster than myoblasts, and the myoblast population is obtained after multiple preplatings [12, 13, 18]. However, the yield of myoblasts is low in conventional preplating techniques, and these techniques fail to completely remove fibroblasts. However, these techniques are not viable for use in cell expansion either due to an incapability of myoblast proliferation or a failure to completely remove fibroblasts. The presence of a small fraction of fibroblasts after purification may significantly reduce myoblast purity during expansion due to fibroblast overgrowth. This study aimed to develop a simple and effective pre-plating technique that not only purifies myoblasts but also maintains their purity during expansion. For this purpose, laminin-coated surfaces, which are known to facilitate myoblast attachment and proliferation [20, 21], were used alone or in-combination with conventional collagen type-I coated surfaces. The yield and purity of myoblasts were evaluated using immunocytochemical staining with myoblast-specific markers to optimize the preplating technique. Purification of several primary cells isolated from human skeletal muscle tissue was also tested to evaluate the effectivity of preplating technique. Moreover, the optimized preplating technique was applied for serial passages to evaluate the maintenance of purity during expansion. 4
Tissue Engineering Part C: Methods One-step purification of human skeletal muscle myoblasts and subsequent expansion using laminin-coated surface (doi: 10.1089/ten.TEC.2015.0015) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
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5
2.
MATERIALS AND METHODS
2.1 Muscle cells harvesting and culture This research was approved by Universiti Kebangsaan Malaysia Research and Ethics Committee (UKMREC) with approval code of FF-037-2013 and FF-313-2010. Cryopreserved primary human skeletal muscle myoblast cells (HSMMs; Lonza, USA) and cells harvested from human skeletal muscle tissue were used in this study. HSMMs of passage 2 were thawed and seeded into a 75-cm2 tissue culture flask with F10:DMEM (1:1, Sigma, USA) containing 10% FBS (PAA Laboratories, Austria), prior to incubation. All of the cell incubations in this study were performed at 37°C with an atmospheric condition of 5% CO2. Waste medium was replaced every 48 hours with fresh culture medium. After reaching 80% confluence, the cells were treated with trypsin-EDTA (Mediatech, USA) for 5 minutes to detach the cells from the culture surface. The cell suspension was then centrifuged and resuspended in fresh culture medium and used for preplating experiments (at passage 4). Human skeletal muscle samples were collected as redundant tissue from four consented patients undergoing lower limb surgery. The samples were processed within 24 hours after surgery. Muscle tissues were clean from fat, connective tissue and blood vessel. Tissue samples were minced into small pieces and digested with 0.25% trypsin (Sigma) in 37°C incubator shaker for 10 minutes. Undigested tissue were separated by centrifugation at 500 rpm for 5 minutes. Supernatant were collected and neutralize with same volume of trypsin inhibitor. This digestion steps were performed for undigested tissue another 2-3 times. Finally, all the supernatant were centrifuge at 1000 rpm for 10 minutes. Pellet was resuspended with F10:DMEM containing 5
Tissue Engineering Part C: Methods One-step purification of human skeletal muscle myoblasts and subsequent expansion using laminin-coated surface (doi: 10.1089/ten.TEC.2015.0015) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
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6 10% FBS. Cells were seeded on 75 cm2 culture flask (Greiner, Germany) and incubate at 37°C with an atmospheric condition of 5% CO2. First medium change was performed 72-96 hours of seeding and subsequent changes were performed every 48 hours. After reaching 80% confluence, the cells were treated with trypsin-EDTA for 5 minutes. The cell suspension was then centrifuged and resuspended in fresh culture medium and used for preplating experiments (at passage 1). 2.2 Immune fluorescence (IF) staining Immune fluorescence staining was performed with anti-desmin and/or anti-fibroblast antibodies to identify myoblasts and fibroblasts, respectively. This technique was also used to evaluate myoblast and fibroblast attachment efficiency as well as myoblast purity and yield. Briefly, adherent cells were fixed with 4% paraformaldehyde (Sigma) and permeabilized with 0.05% triton X-100 (Sigma). After masking the nonspecific proteins with 10% goat serum (Gibco, USA), the cells were incubated for 1 h at room temperature with a mixture of rabbit antihuman desmin (1:250; Novus Biologicals, USA) and mouse anti-fibroblast clone TE-7 (1:100; Millipore, USA). Cells were then immunolabeled with a mixture of Alexa Fluor 594 goat antirabbit and Alexa Fluor 488 goat anti-mouse (1:250; Molecular Probes; USA), followed by counter staining with dye 4'-6-diamidino-2-phenylindole (DAPI; Molecular Probes). HSMMs and primary cells isolated from skeletal muscle tissue were also stained only for the anti-human desmin antibody using a similar protocol. At least five images were captured randomly from each plate using a Nikon A1 confocal microscope. Cells were manually quantified for total myoblasts (Desmin positive), total fibroblasts (fibroblast marker positive) and total cells (DAPI positive). In the case of single staining with Desmin, the total number of fibroblasts was calculated by subtracting the number of total myoblasts from the total number of cells. 6
Tissue Engineering Part C: Methods One-step purification of human skeletal muscle myoblasts and subsequent expansion using laminin-coated surface (doi: 10.1089/ten.TEC.2015.0015) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
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7 2.3 Surface coating with laminin and collagen type I Surface coating with laminin (Sigma) has been described elsewhere [21]. Briefly, 50 g/ml of laminin solution was added to the culture surface and incubated for 1-2 hours. Culture surfaces were washed with pre-warmed PBS (Sigma) prior to cell culture use. Rat tail collagen type I (Sigma) was used to coat the culture surface according to the manufacturer's protocol. Briefly, the culture surface was incubated with 250g/ml collagen type I for 2-3 hours. Excess fluid was then removed, and the culture flask was dried overnight at ambient temperature. The culture surface was washed with pre-warmed PBS prior to cell culture use. 2.4 Preplating Figure 1 demonstrates the preplating experiment on different orders of laminin- and collagen type I-coated surfaces. For the serial plating experiment, 2.5×103 cells/cm2 were seeded on the 1st plate (collagen type I- or laminin-coated surface) and incubated for 5, 10 or 15 minutes. Unattached cells were then transferred to the 2nd plate (collagen type I- or laminincoated surface) and similarly incubated either for 5, 10 or 15 minutes. Finally, the rest of the unattached cells were transferred onto the 3rd plate (plain surface). The attached cells after serial preplating on different surfaces were incubated for 24 hours prior to immunofluorescence (IF) staining. To evaluate the myoblast purity of cells that were used for preplating, the cells were seeded onto a plain surface without serial plating and incubated for 24 hours prior to IF staining (control). Preplating experiment with primary cells were performed only for the best condition to confirm the effectivity of the purification techniques. The purity and yield of myoblasts for each plate of preplating were evaluated using the following equations: 7
Tissue Engineering Part C: Methods One-step purification of human skeletal muscle myoblasts and subsequent expansion using laminin-coated surface (doi: 10.1089/ten.TEC.2015.0015) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
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8 Purity of myoblasts
Yield of myoblasts
No. of myoblasts on plate of interest 100% Total cells on the plate of interest
No. of myoblasts on plate of interest 100% Total no. of myoblasts on all 3 plates of preplating
2.5 Attachment efficiency Attachment efficiency of myoblasts and fibroblasts was evaluated on plain, collagen type Iand laminin-coated surface for the incubation period of 5, 10 or 15 minutes using preplating experiment as described in previous section. The conditions were listed in Fig 1. Efficiency of myoblast and fibroblast attachment was evaluated only at the 1st plate of serial pre-plating. Briefly, mixed population of myoblasts and fibroblasts were seeded on different culture surface (1st plate), and incubated for 5, 10 or 15 minutes to facilitate the attachment of myoblasts and fibroblasts. Unattached cells were transferred to subsequent plates as shown in Fig 1. Finally, adherent cells were incubated for another 24 hours, and IF staining was perform to evaluate number of attached myoblasts and fibroblasts (as describe in section 2.2). The efficiency of myoblast and fibroblast attachment for 5, 10 or 15 minutes incubation on different culture surface was evaluated via following equation: No. of myoblasts at 1st plate of preplating Total no. of myoblasts on 3 plates of preplating No. of fibroblast s at 1st plate of preplating Fibroblast attachment efficiency for incubation time t, Af, t Total no. of fibroblast s on 3 plates of preplating
Myoblast attachment efficiency for incubation time t, Am, t
2.6 Purification and subsequent cell expansion To evaluate the maintenance of myoblast purity during cell expansion, purification and expansion of myoblasts were performed on laminin-coated surface for 2 subsequent passages. Purification was performed via preplating of mixed cells on laminin-coated surface for 5 minutes, and attached cells on laminin-coated surface was expanded for 144 hours. Finally, cells 8
Tissue Engineering Part C: Methods One-step purification of human skeletal muscle myoblasts and subsequent expansion using laminin-coated surface (doi: 10.1089/ten.TEC.2015.0015) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
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9 were trypsinize and pre-plated onto laminin-coated surface for 5 minutes to perform another round of purification. Myoblast purity was evaluated at 0 hour (initial sample without purification), 24 hours (after 1st purification), 144 hours (after trypsinization without purification) and 168 hours (24 hours after 2nd purification) using protocol described in earlier sections. Moreover, growth rate of myoblasts and fibroblasts was also evaluated on laminin-coated surface along with that on plain surface. For this purpose, mixed population of cells were seeded on plain and laminin-coated surface at seeding density of 2.5×103 cells/cm2 and culture for 144 hours. Adherent cells at 24 and 144 hours were stained with anti-desmin and anti-fibroblasts (as described earlier) to determine the myoblasts and fibroblasts population, respectively. The growth rate of myoblasts and fibroblasts were evaluated using the following equationGrowth rate (h-1) = Ln (cell concentration at 144 h / cell concentration at 24 h) /120 h 2.8 Statistical Analysis All the experiment were performed in triplicate. The value was shown as mean ± standard deviation. Student’s t-tests were performed to determine statistical significance. P value less than 0.05 was considered as statistically significant.
3.
RESULTS
3.1 Identification of myoblasts and fibroblasts Myoblasts and fibroblasts in a mixed culture were identified via immunostaining as an invasive technique. Only two types of cells were identified in culture (Fig. 2). A group of cells 9
Tissue Engineering Part C: Methods One-step purification of human skeletal muscle myoblasts and subsequent expansion using laminin-coated surface (doi: 10.1089/ten.TEC.2015.0015) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
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10 was positive for only desmin and identified as myoblasts, whereas the other group was positive for only the fibroblast-specific marker and identified as fibroblasts. On the basis of this observation, for further experiments in this study, immunostaining was employed only with desmin, and desmin-positive cells were identified as myoblasts and desmin-negative cells were identified as fibroblasts. 3.2 Attachment efficiency of myoblasts and fibroblasts To evaluate the efficiency of myoblast and fibroblast attachment on plain, laminin- and collagen type I-coated surfaces in relationship to time, a mixed population of myoblasts and fibroblasts was seeded onto the designated culture surface and incubated for 5, 10 and 15 minutes. Representative images from each condition are shown in Figure 3A. It was found that myoblasts preferentially attached to the laminin-coated surface and that the efficiency of myoblast attachment (Am) on laminin-coated surfaces was significantly higher for an incubation period of 5, 10 and 15 minutes compared to fibroblasts (Fig. 3B). The attachment of myoblasts onto the laminin-coated surface was initiated immediately as the Am value for the 5-minute incubation was 0.58±0.06, which increased significantly to 0.81±0.02 for the 10-minute incubation. The value of Am for the 15-minute incubation showed a further increase (0.85±0.05) but was not significantly different from that of 10 minutes. It was also found that the efficiency of fibroblast attachment (Af) on laminin-coated surfaces increased significantly with an increase in incubation time and that the rate of the increase was comparatively higher compared to myoblasts. Fibroblast attachment on laminin-coated surface for 10 and 15 minutes incubation increased 2.8 and 3.6 folds compared to 5 minutes incubation, respectively, whereas for myoblasts increased 1.4 and 1.5 folds, respectively.
10
Tissue Engineering Part C: Methods One-step purification of human skeletal muscle myoblasts and subsequent expansion using laminin-coated surface (doi: 10.1089/ten.TEC.2015.0015) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
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11 In contrast to myoblasts, fibroblasts preferentially attached onto collagen type I-coated surfaces, and the Af on collagen type I-coated surfaces for 10- and 15-minute incubations was significantly higher compared to Am, although no significant differences were observed for the 5minute incubation (Fig. 3). The Af value on the collagen type I-coated surface increased sharply from 0.25±0.11 at 5 minutes of incubation to 0.78±0.08 at 10 minutes of incubation. No further increase was detected for the 15-minute incubation period. On a plain surface, the efficiency of myoblast and fibroblast attachment also increased with an increase in incubation time; however, no significant difference was observed for myoblast and fibroblast attachment. 3.3 Efficiency of myoblast purification To purify myoblasts, the serial plating technique was employed using HSMMs, as demonstrated schematically in Fig. 1. The quantitative evaluation for purity is demonstrated in Figure 4A. The highest purity of myoblasts was achieved by incubating the mixed population of cells on the laminin-coated surface for 5 minutes (1st plate of serial plating), and the purity was estimated to be ~79%, which was 1.7 times higher compared to the initial population and was statistically significant. However, the purity of myoblasts significantly decreased after 10- and 15-minute incubations on the laminin-coated surface (1st plate of serial plating). High purity myoblast populations were also obtained from two other conditions. In one case, two subsequent plating on collagen-coated surfaces for 10 minutes resulted in a myoblast purity of ~71% on a subsequent plate, i.e., on a plain surface. In another case, the cells incubated for 15 minutes first on the collagen type I-coated surface and then subsequently on the laminin-coated surface resulted in a purity of ~74% on the laminin-coated surface.
11
Tissue Engineering Part C: Methods One-step purification of human skeletal muscle myoblasts and subsequent expansion using laminin-coated surface (doi: 10.1089/ten.TEC.2015.0015) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
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12 To understand the effectiveness of the purification process, the yield of myoblasts in each condition was also evaluated (Fig. 4B). Yield of myoblasts on the laminin-coated surface (1st plate of serial plating) after the 5-minute incubation was ~57%. However, the yields of myoblasts on the laminin-coated surface with 10- and 15-minute incubations were 1.4 and 1.3 times higher compared to the 5-minute incubation, respectively (p
