- Email: [email protected]
New isolation system for collecting living cells from tissue
Journal of Bioscience and Bioengineering VOL. 115 No. 1, 100e103, 2013 www.elsevier.com/locate/jbiosc
TECHNICAL NOTE
New isolation system for collecting living cells from tissue Takahiro Shioyama,1, 2 Yuji Haraguchi,1 Yoshihiro Muragaki,1 Tatsuya Shimizu,1 and Teruo Okano1, * Institute of Advanced Biomedical Engineering and Science, TWIns, Tokyo Women’s Medical University, 8-1 Kawada-cho, Shinjuku-ku, Tokyo 162-8666, Japan1 and Nihon Kohden Corporation, 1-31-4 Nishiochiai, Shinjuku-ku, Tokyo 161-8560, Japan2 Received 10 July 2012; accepted 17 August 2012 Available online 10 October 2012
A new semi-automatic living-cell isolation system was developed. The new system improves the quality of isolated cells, reduces cell isolation time, and isolates more cells with a higher cell viability compared to conventional manual methods. We successfully applied this system to isolate beating cardiomyocytes and fabricate electrical communicative cardiac tissue. In this study using the isolated cardiac cells we also fabricated a cardiac cell sheet that beat spontaneously and synchronously. Ó 2012, The Society for Biotechnology, Japan. All rights reserved. [Key words: Automated cell isolation; Primary culture; Cardiac cells; Heart tissue; Tissue engineering]
Cell-based therapy and tissue engineering have been recognized as the most promising methods for curing tissue/organ failure (1). Clinical trials of direct cell injection and the transplantation of tissues produced by tissue engineering have already been performed in various tissues (2e5). In those cell therapies, functional cells are isolated from healthy tissues or organs, and are then proliferated by in vitro culture. The cells or tissues fabricated by tissue-engineering methods can then be transplanted to damaged tissues for regeneration and functional repair. In some cases, the isolation of cells from tissues/organs was performed with enzyme treatment after manual mincing with a surgical knife [e.g., cardiac cells (6), adipose tissuederived cells and mesenchymal stem cells (7,8), kidney proximal tubule epithelial cells (9), skeletal muscle myoblasts (10), thyroid cells (11), chondrocytes (12), and endometrial stromal cells (13)]. The degree of success and quality of cells isolated by all manual isolation methods depends largely on the skill and experience of the technician. The tedious manipulations required increases the possibility of human error, including infectious contamination. Therefore, automating these manipulations will improve the quality of cells that are required for advanced tissue engineering and make research results more reproducible. In this paper, a new semi-automated cell isolation system was able to mince tissues/organs mechanically with the help of enzymes, and the variation of results between experienced and inexperience technicians was substantially narrowed. For this study beating cardiac cells were semi-automatically isolated from rat hearts by the system, and we believe that cells of many other tissues can also be successfully isolated. All protocols were performed according to the Guideline of Tokyo Women’s Medical University on Animal Use, The Principles
* Corresponding author. Tel.: þ81 3 5367 9945x6201; fax: þ81 3 3359 6046. E-mail address: [email protected] (T. Okano).
of Laboratory Animal Care formulated by the National Society for Medical Research, and the Guide for the Care and Use of Laboratory Animals prepared by the Institute of Laboratory Animal Resources and published by the National Institutes of Health (NIH Publication No. 86-23, revised 1985). A cell-isolating system was developed (Fig. 1). The system has three parts: a stirring device, with four knives (Fig. 1A), a chamber (Fig. 1B), and a rotation controller. The stirring device was assembled with a rotation controller before use (Fig. 1). The chamber was designed with a computer-aided design system (InventorⓇ, Autodesk, San Rafael, CA, USA), and the data were transferred to a rapid prototyping system (EDEN350Ô, Objet Geometries, Rehovot, Israel). The chamber was made from a photopolymer (FullCureⓇ720, Objet Geometries). The motor for the stirring device rotates intermittently (Movie S1). The isolation of cardiac cells using this new system was performed as follows; (i) isolated ventricles from neonatal SD rats (Nisseizai, Tokyo, Japan) were put in the device chamber after measuring the total weight of the ventricles; (ii) pre-warmed (37 C) Hanks’ balanced salts solution (SigmaeAldrich Japan, Tokyo) containing 1.5 mg/L collagenase (type II) (Worthington Biochemical, Lakewood, NJ, USA) was added to the chamber; (iii) the motor was rotated at various speeds with the temperature at 37 C; (iv) after being minced by the stirring device and filtered through a membrane filter (pore size: 40 mm) to remove the tissue residue, the cell suspensions were put in a 50 mL tube containing an equal volume of Hanks’ balanced salts solution with 50% fetal bovine serum (FBS) and were centrifuged (1700 g for 5 min at 4 C); (v) the collected cells were suspended in a culture medium composed of 6% FBS, 40% Medium 199 (Invitrogen, Carlsbad, CA, USA), 0.2% penicillin-streptomycin solution, 2.7 mmol/L glucose, and 54% balanced salt solution containing (in mmol/L) 116 NaCl, 1.0 NaH2PO4, 0.8 MgSO4, 1.18 KCl, 0.87 CaCl2, and 26.2 NaHCO3.
1389-1723/$ e see front matter Ó 2012, The Society for Biotechnology, Japan. All rights reserved. http://dx.doi.org/10.1016/j.jbiosc.2012.08.013
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FIG. 1. A new cell isolation system. The cell isolation device has three parts: a stirring device (A), which has four knives, a chamber (B), and a rotation controller (C). The chamber was designed with a computer-aided design system and the data were transferred to a rapid prototyping system. The motor for the stirring device is able to rotate intermittently (Movie S1).
A conventional manual method was also used for comparison, where the isolation of rat cardiac cells was performed according to a previously report (14). Briefly, ventricles were manually minced with a surgical knife and incubated in Hanks’ balanced salts solution containing collagenase in an Erlenmeyer flask that was put in a shaking water-bath at 37 C for 50 min. Static culture was performed for 10 min, and then shaking culture (130 rpm) was performed for 40 min. During the shaking culture, the collagenase solution was exchanged with new collagenase solution every 10 min. After being filtered through a membrane filter (pore size: 40 mm), the cell suspensions were put in a 50 mL tube containing an equal volume of Hanks’ balanced salts solution with 50% FBS and centrifuged (1700 g for 5 min at 4 C). The collected cells were suspended in the same culture medium described above. The isolated cells were counted by a counting chamber after treatment with a DNA-binding dye, Hoechst 33342 (Invitrogen). Only nuclear positive cells were counted to avoid contamination with erythrocytes. Microscopic photographs of cardiac cells were observed by a phase-contract microscope (ET300, Nikon, Tokyo), and the images were recorded by a digital video camera (DCR-TRV900, Sony, Tokyo) through a CCD camera (HV-D28S, Nikon). Isolated cells were plated onto a 4-well culture dish at a concentration of 6.0 105 cells/well. After being cultured for 4 days, the cultured cells were fixed with 4% paraformaldehyde and permeabilized with 0.15% Triton X-100 in phosphate-buffered saline; after which, indirect immunofluorescence assays were performed. Cardiac troponin T antibody (mouse monoclonal antibody, Funakoshi, Tokyo) and Alexa-Fluor 488-labeled anti-mouse IgG antibody (Invitrogen) were used as the primary and secondary antibodies, respectively. Prepared specimens were examined by a fluorescence microscope (ELIPSE TE2000-U, Nikon) with a CCD camera (Axio Cam HRc, Carl Zeiss, Hallbergmoos, Germany). Creatine kinase (CK) is an enzyme normally found in cardiomyocytes so that CK activity can be used as an index for estimating the relative ratio of cardiomyocytes per isolated cells. After being centrifuged, the collected cells (6.0 105 cells) were lysed with 0.15% Triton X-100, and CK activity was measured by an enzymatic method at an outsourcing laboratory, SRL (Tokyo). CK activity was measured per 6.0 105 of isolated cells. Normally existing in the soluble fractions of cells, when CK is found in blood it indicates cell injury (14,15). Therefore, CK activity measurement can indicate the viability of cardiomyocytes after cell isolation. After 4 days of cultivation, the CK activity in both the cells and in the culture supernatants was measured. The CK activity in the cardiomyocytes was measured after treatment with 0.15% Triton X-100. The viability of cardiomyocytes was calculated by the following equation;
Relative CK activityð%Þ ¼
CKact-cell 100 CKact-cell þ CKact-medium
(1)
CKact-cell and CKact-medium are CK activities in the cells and in the culture supernatants, respectively. In addition, cell viability was also measured by the LIVE/DEAD Viability/Cytotoxicity Kit (Invitrogen), which contains calcein AM and ethidium homodimer-1. Calcein AM, which is a membrane-permeant reagent, is cleaved by esterases in live cells to yield cytoplasmic green fluorescent dye, and membrane-impermeant ethidium homodimer-1 is inserted into the nucleic acids of membrane-injured cells with red fluorescence. Briefly, isolated cardiac cells were cultured for 4 days and cells were stained with calcein AM and ethidium homodimer-1 for 30 min at 37 C. The fluorescent images of the cells were photographed by a fluorescence microscope (ECLIPSE TE2000-U) with a CCD camera (Axio Cam HRc, Carl Zeiss). The number of isolated cells per weight of heart tissue, CK activity per unit number of isolated cells, and the viability of cardiomyocytes that were isolated by both methods were examined. The mean and standard deviation of the data from each individual technician were obtained, and an unpaired Student’s t-test was performed to compare both methods. Experimental errors of the new system and the conventional method were estimated by the coefficient of variation (CV). CV was calculated by the following equation: CV ¼
Standard deviations 100 Means
(2)
A p-value of less than 0.01 was considered significant. The isolated cardiac cells were plated onto a 35 mm diameter temperature-responsive culture dish (UpcellⓇ) (CellSeed, Tokyo) at a concentration of 2.4 106 cells/dish. After 4 days, to release the confluent cells as an intact sheet, the culture dishes were placed in a CO2 incubator set at 20 C. The obtained cell sheet was fixed with 4% paraformaldehyde and embedded in paraffin, sectioned, and stained with hematoxylin and eosin. Prepared specimens were examined by a microscope (ELIPSE E800, Nikon). In the new method, a rotation speed of 200 rpm for 30 min with an interruption (0.5-s rotation and 1-s interruption) (Movie S1) was used for the following reasons. (i) The ratio of cardiac troponin Tpositive cells after incubation for 4 days decreased by increasing the rotation speed from 200 rpm to 500 rpm (Fig. 2AeD). (ii) Similar results were obtained in CK activity for a unit number of isolated cells (data not shown). In addition, cardiac cells isolated with a rotation speed of 500 rpm beat only partly, while cardiac cells isolated with a rotation speed of 200 rpm beat completely and synchronously (Movies S2 and S3). (iii) When a rotation speed of 100 rpm was used, the number of isolated cells was less than at
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FIG. 2. Immunocytochemistry of cardiac cells in the isolated cells. The cells were isolated for 15 min (AeD) and for 30 min (E). Rotation speed: (A, E) 200 rpm; (B) 300 rpm; (C) 400 rpm; and (D) 500 rpm. In the conventional manual method cells were incubated for 50 min (F). After isolated cells were counted, these cells were plated onto a 4-well culture dish at a concentration of 6.0 105 cells/well. Cardiac troponin T in the cultured cardiomyocytes was stained green with a cardiac-specific troponin T antibody. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
200 rpm (data not shown). (iv) The number of isolated cells increased time-dependently up to 30 min (data not shown). The number of isolated cells using interrupted rotation was greater than with continuous rotation (data not shown). The numbers of isolated cells per weight of heart tissue by the new method (rotation of 200 rpm for 30 min) and the conventional method (incubation for 50 min) were (9.7 1.8) 104 cells/mg and (5.9 1.3) 104 cells/ mg, respectively (Fig. 3A). The difference between the results was statistically significant. CK activities per unit number of isolated cells by the new method and the conventional method were 488.4 25.0 U/1.2 106 cells and 478.4 108.5 U/1.2 106 cells, respectively (Fig. 3B). The viabilities of cardiomyocytes isolated by the new method and the conventional method were 98.5 0.3% and 95.5 1.3%, respectively (Fig. 3C). Similar results were shown by LIVE/DEAD assay, namely, cells isolated in both cell isolation systems largely lived (Fig. S1). Here again the difference between the two methods was statistically significant. These results showed that the number of isolated cells and the viability of cardiomyocytes improved using the new method, while the relative ratio of cardiomyocytes was almost equal. The number of cardiac troponin T-positive cells after incubation for 4 days isolated at 200 rpm for 30 min were almost equal to that of the conventional method (Fig. 2E and F). In addition, the CV of the number of isolated cells, the CK activity, and the viability of cardiomyocytes from the new system were less than those of the conventional method. In
J. BIOSCI. BIOENG., particular the CV of CK activity, and the viability of cardiac cells with the new system were extremely low (Table S1). These results show that the reproducibility of data from experiments by the new method was higher than with the conventional method. This supports the premise that the new semi-automatic system reduces individual variation of results in the isolation of living cells, which in turn will produce better results in tissue-engineering applications that rely on a consistent source of living cells. To confirm the actual beating function of cardiomyocytes, the isolated cells were carefully observed. Spontaneous beating was observed in isolated cardiac cells at least 1 day after the seeding onto culture dishes, and these cells then gradually developed synchronous beating (Movie S2). These results show that functional and living cardiomyocytes could be isolated by the new method just like the conventional method. There are several reports regarding methods for cardiac cell isolation (6,16), and here we present a number of significant improvements in a new cell isolation system. Cardiomyocytes having the potential to beat were isolated from neonatal rat hearts by using this new isolation system. In this study, the optimal conditions were examined in detail; namely, optimal rotation speed, condition, and the duration of rotation. The optimal conditions increased the efficiency of cell isolation. Consequently, the new system was able to isolate approximately 1.6 times more cells than the conventional manual method, while the isolation time of the new method was almost half that of the conventional method (Fig. 3). The new system also increased the viability of cardiomyocytes (Fig. 3). Therefore, the new isolation system is thought to provide a significant improvement in cell isolation for use in regenerative medicine. In our cell isolation system, 200 rpm was the optimal speed for isolating beating cardiomyocytes from heart tissues. On the other hand, to isolate cells from other tissues, the optimal conditions of the new isolation system could vary depending on target cell type. Recently, several reports about automatic cell culture systems have been published (17,18). Automation that removes manual manipulation has been advanced in the field of tissue-engineering and regenerative medicine, because manual manipulation increases the possibility of human error, including infectious contamination. In this report, the mincing of tissues/organs and cell isolation with enzymes were automated to avoid possible adverse effects due to variations of individual technicians which could affect the viabilities, conditions, and proportions of the isolated cells. In fact, the CVs of all items in the conventional manual method were larger than those in the new method (Table S1). Although, the difference in the CVs of the relative ratio of cardiac cells and the relative CK activity between the two methods was large, the difference in the CV of the number of isolated cells was small (Table S1). Variation in cell density of individual tissues, which is beyond the control of our isolation system, might induce variation in the number of isolated cells. Constancy in the CVs of the relative ratio of cardiac cells and the viabilities of cardiac cells should contribute to more consistent quality of isolated cells, but not the number of isolated cells. Consistency of cell quality is important for tissue-engineering and regenerative medicine, which makes our isolation system an important improvement in the development of tissue-engineering and regenerative medicine. At present our system requires pretreatment to isolate homogenous tissue before cell isolation. In the case of complex tissue, additional pretreatment is necessary before cell isolation. For example, in the isolation of adipose tissue-derived stem cells, small vessels and fibrous tissue should first be removed from the adipose tissue. Further developments of a cell isolation system might require the addition of robot technology, for example, to establish a truly automated cell isolation system that includes the isolation of homogenous tissue. In the case of using a temperature-responsive culture dish, when culture temperature was decreased from 37 C to 20 C, cardiac cells detached themselves as a contiguous cell sheet within 1 h. The
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FIG. 3. Effect of the new system and the conventional method on the number of isolated cells per weight of heart tissue, the relative ratio of cardiomyocytes, and the relative creatine kinase activity. Three persons independently performed each experiment. A rotation of 200 rpm for 30 min with an interruption rotation (0.5-s rotation and 1-s interruption) was used in the new method. The number of isolated cells per weight of heart tissue (A), the relative CK activity per isolated cells (B) and the relative CK activity (C) were measured as followed: Nuclear positive cell numbers ðcellsÞ The number of isolated cells per weight of the heart tissues ¼ Total weight of heart tissues ðmgÞ Creatine kinase activity ðUÞ The relative ratio of cardiomyocytes per the isolated cells ¼ 1:2 106 nuclear positive cells CKact-cell The relative CK activityð%Þ ¼ 100 CKact-cell þ CKact-medium where CKact-cell and CKact-medium are CK activities in the cells and CK activities in the culture supernatants, respectively. The data are expressed as mean standard deviation (**p < 0.01, n ¼ 5).
released cardiac cell sheets shrank due to cytoskeletal tensile reorganization (Fig. S2A) (the area of sheet: approximately 9.6 cm2 / 2.4 cm2), resulting in a double- or triple-layered cell sheet. The thickness of the cell sheet was found to be approximately 20e30 mm (Fig. S2B). The harvested cell sheet continued to show a synchronous and spontaneous beating even after detachment (Movie S4). These results showed that an electrical communicative and functional cardiac cell sheet could be fabricated from cardiac cells isolated by the new method, just like the conventional method. In conclusion, a new-automated system was developed to isolate living cells from tissue/organs and was demonstrated by isolating beating cardiac cells from heart tissue. Our new system isolated higher quality cells and reduced cell isolation time while also improving the viability of the cells. All current and future applications in regenerative medicine are dependent on a consistent and reliable supply of high quality, viable living cells. We are confident that this new isolation system can be a powerful new tool for use in the fields of bioengineering, tissue-engineering and regenerative medicine. Supplementary data related to this article can be found online at http://dx.doi.org/10.1016/j.jbiosc.2012.08.013. We appreciate the useful comments and technical criticism from Prof. Hiroshi Iseki and Dr. Norio Ueno (Institute of Advanced Biomedical Engineering and Science, Tokyo Women’s Medical University). As well as the support and encouragement of Prof. Sunao Takeda (He was with Nihon Kohden and is now with the Dept. of Clinical Engineering, Tokyo Univ. of Technology, Tokyo, Japan), Shinji Yamamori, Hirotsugu Kubo and Akane Suzuki (Nihon Kohden). This work is granted by the Japan Society for the Promotion of Science (JSPS) through the “Funding Program for WorldLeading Innovative R&D on Science and Technology (FIRST Program),” initiated by the Council for Science and Technology Policy (CSTP) and is supported by grants from the Global Center of Excellence (GCOE) Program, Multidisciplinary Education and Technology and Research Center for Regenerative Medicine (MERCREM) from the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan.
References 1. Atala, A., Lanza, R., Thomson, J. A., and Nerem, R. (Eds.): Principles of regenerative medicine, 2nd ed. Academic Press (2011). 2. Chachques, J. C.: Cellular cardiac regenerative therapy in which patients? Expert Rev. Cardiovasc. Ther., 7, 911e919 (2009). 3. Lee, K., Chan, C. K., Patil, N., and Goodman, S. B.: Cell therapy for bone regeneration e bench to bedside, J. Biomed. Mater. Res. B Appl. Biomater., 89, 252e263 (2009).
4. van Osch, G. J., Brittberg, M., Dennis, J. E., Bastiaansen-Jenniskens, Y. M., Erben, R. G., Konttinen, Y. T., and Luyten, F. P.: Cartilage repair: past and future e lessons for regenerative medicine, J. Cell. Mol. Med., 13, 792e810 (2009). 5. Tedesco, F. S., Dellavalle, A., Diaz-Manera, J., Messina, G., and Cossu, G.: Repairing skeletal muscle: regenerative potential of skeletal muscle stem cells, J. Clin. Invest., 120, 11e19 (2010). 6. Shimizu, T., Yamato, M., Isoi, Y., Akutsu, T., Setomaru, T., Abe, K., Kikuchi, A., Umezu, M., and Okano, T.: Fabrication of pulsatile cardiac tissue grafts using a new 3-dimensional cell sheet manipulation technique and temperatureresponsive cell culture surfaces, Circ. Res., 90, e40ee48 (2002). 7. Miyahara, Y., Nagaya, N., Kataoka, M., Yanagawa, B., Tanaka, K., Hao, H., Ishino, K., Ishida, H., Shimizu, T., Kangawa, K., and other 4 authors: Monolayered mesenchymal stem cells repair scarred myocardium after myocardial infarction, Nat. Med., 12, 459e465 (2006). 8. Toyoda, M., Matsubara, Y., Lin, K., Sugimachi, K., and Furue, M.: Characterization and comparison of adipose tissue-derived cells from human subcutaneous and omental adipose tissues, Cell Biochem. Funct., 27, 440e447 (2009). 9. Hara-Chikuma, M. and Verkman, A. S.: Aquaporin-1 facilitates epithelial cell migration in kidney proximal tubule, J. Am. Soc. Nephrol., 17, 39e45 (2006). 10. Kondoh, H., Sawa, Y., Fukushima, N., Matsumiya, G., Miyagawa, S., KitagawaSakakida, S., Imanishi, Y., Kawaguchi, N., Matsuura, N., and Matsuda, H.: Combined strategy using myoblasts and hepatocyte growth factor in dilated cardiomyopathic hamsters, Ann. Thorac. Surg., 84, 134e141 (2007). 11. Arauchi, A., Shimizu, T., Yamato, M., Obata, T., and Okano, T.: Tissue-engineered thyroid cell sheet rescued hypothyroidism in rat models after receiving total thyroidectomy comparing with nontransplantation models, Tissue Eng. Part A, 15, 3943e3949 (2009). 12. He, X., Lu, H., Kawazoe, N., Tateishi, T., and Chen, G.: A novel cylinder-type PLLA-collagen hybrid sponge for cartilage, Tissue Eng. Part C, 16, 329e338 (2010). 13. Matsumoto, K., Yamauchi, N., Watanabe, R., Oozono, S., Kubota, K., Nishimura, K., Wood, C., Soh, T., Kizaki, K., and Hattori, M. A.: In vitro decidualization of rat endometrial stromal cells, Cell Tissue Res., 335, 575e583 (2009). 14. Brancaccio, P., Maffulli, N., and Limongelli, F. M.: Creatine kinase monitoring in sport medicine, Br. Med. Bull., 81/82, 209e230 (2007). 15. Schaser, K. D., Bail, H. J., Schewior, L., Stover, J. F., Melcher, I., Haas, N. P., and Mittlmeier, T.: Acute effects of N-acetylcysteine on skeletal muscle microcirculation following closed soft tissue trauma in rats, J. Orthop. Res., 23, 231e241 (2005). 16. Kinugawa, K., Shimizu, T., Yao, A., Kohmoto, O., Serizawa, T., and Takahashi, T.: Transcriptional regulation of inducible nitric oxide synthase in cultured neonatal rat cardiac myocytes, Circ. Res., 81, 911e921 (1997). 17. Kino-oka, M. and Taya, M.: Recent developments in processing systems for cell and tissue cultures toward therapeutic application, J. Biosci. Bioeng., 108, 267e276 (2009). 18. Kato, R., Iejima, D., Agata, H., Asahina, I., Okada, K., Ueda, M., Honda, H., and Kagami, H.: A compact, automated cell culture system for clinical scale cell expansion from primary tissues, Tissue Eng. Part C, 16, 947e956 (2010).
TECHNICAL NOTE
New isolation system for collecting living cells from tissue Takahiro Shioyama,1, 2 Yuji Haraguchi,1 Yoshihiro Muragaki,1 Tatsuya Shimizu,1 and Teruo Okano1, * Institute of Advanced Biomedical Engineering and Science, TWIns, Tokyo Women’s Medical University, 8-1 Kawada-cho, Shinjuku-ku, Tokyo 162-8666, Japan1 and Nihon Kohden Corporation, 1-31-4 Nishiochiai, Shinjuku-ku, Tokyo 161-8560, Japan2 Received 10 July 2012; accepted 17 August 2012 Available online 10 October 2012
A new semi-automatic living-cell isolation system was developed. The new system improves the quality of isolated cells, reduces cell isolation time, and isolates more cells with a higher cell viability compared to conventional manual methods. We successfully applied this system to isolate beating cardiomyocytes and fabricate electrical communicative cardiac tissue. In this study using the isolated cardiac cells we also fabricated a cardiac cell sheet that beat spontaneously and synchronously. Ó 2012, The Society for Biotechnology, Japan. All rights reserved. [Key words: Automated cell isolation; Primary culture; Cardiac cells; Heart tissue; Tissue engineering]
Cell-based therapy and tissue engineering have been recognized as the most promising methods for curing tissue/organ failure (1). Clinical trials of direct cell injection and the transplantation of tissues produced by tissue engineering have already been performed in various tissues (2e5). In those cell therapies, functional cells are isolated from healthy tissues or organs, and are then proliferated by in vitro culture. The cells or tissues fabricated by tissue-engineering methods can then be transplanted to damaged tissues for regeneration and functional repair. In some cases, the isolation of cells from tissues/organs was performed with enzyme treatment after manual mincing with a surgical knife [e.g., cardiac cells (6), adipose tissuederived cells and mesenchymal stem cells (7,8), kidney proximal tubule epithelial cells (9), skeletal muscle myoblasts (10), thyroid cells (11), chondrocytes (12), and endometrial stromal cells (13)]. The degree of success and quality of cells isolated by all manual isolation methods depends largely on the skill and experience of the technician. The tedious manipulations required increases the possibility of human error, including infectious contamination. Therefore, automating these manipulations will improve the quality of cells that are required for advanced tissue engineering and make research results more reproducible. In this paper, a new semi-automated cell isolation system was able to mince tissues/organs mechanically with the help of enzymes, and the variation of results between experienced and inexperience technicians was substantially narrowed. For this study beating cardiac cells were semi-automatically isolated from rat hearts by the system, and we believe that cells of many other tissues can also be successfully isolated. All protocols were performed according to the Guideline of Tokyo Women’s Medical University on Animal Use, The Principles
* Corresponding author. Tel.: þ81 3 5367 9945x6201; fax: þ81 3 3359 6046. E-mail address: [email protected] (T. Okano).
of Laboratory Animal Care formulated by the National Society for Medical Research, and the Guide for the Care and Use of Laboratory Animals prepared by the Institute of Laboratory Animal Resources and published by the National Institutes of Health (NIH Publication No. 86-23, revised 1985). A cell-isolating system was developed (Fig. 1). The system has three parts: a stirring device, with four knives (Fig. 1A), a chamber (Fig. 1B), and a rotation controller. The stirring device was assembled with a rotation controller before use (Fig. 1). The chamber was designed with a computer-aided design system (InventorⓇ, Autodesk, San Rafael, CA, USA), and the data were transferred to a rapid prototyping system (EDEN350Ô, Objet Geometries, Rehovot, Israel). The chamber was made from a photopolymer (FullCureⓇ720, Objet Geometries). The motor for the stirring device rotates intermittently (Movie S1). The isolation of cardiac cells using this new system was performed as follows; (i) isolated ventricles from neonatal SD rats (Nisseizai, Tokyo, Japan) were put in the device chamber after measuring the total weight of the ventricles; (ii) pre-warmed (37 C) Hanks’ balanced salts solution (SigmaeAldrich Japan, Tokyo) containing 1.5 mg/L collagenase (type II) (Worthington Biochemical, Lakewood, NJ, USA) was added to the chamber; (iii) the motor was rotated at various speeds with the temperature at 37 C; (iv) after being minced by the stirring device and filtered through a membrane filter (pore size: 40 mm) to remove the tissue residue, the cell suspensions were put in a 50 mL tube containing an equal volume of Hanks’ balanced salts solution with 50% fetal bovine serum (FBS) and were centrifuged (1700 g for 5 min at 4 C); (v) the collected cells were suspended in a culture medium composed of 6% FBS, 40% Medium 199 (Invitrogen, Carlsbad, CA, USA), 0.2% penicillin-streptomycin solution, 2.7 mmol/L glucose, and 54% balanced salt solution containing (in mmol/L) 116 NaCl, 1.0 NaH2PO4, 0.8 MgSO4, 1.18 KCl, 0.87 CaCl2, and 26.2 NaHCO3.
1389-1723/$ e see front matter Ó 2012, The Society for Biotechnology, Japan. All rights reserved. http://dx.doi.org/10.1016/j.jbiosc.2012.08.013
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FIG. 1. A new cell isolation system. The cell isolation device has three parts: a stirring device (A), which has four knives, a chamber (B), and a rotation controller (C). The chamber was designed with a computer-aided design system and the data were transferred to a rapid prototyping system. The motor for the stirring device is able to rotate intermittently (Movie S1).
A conventional manual method was also used for comparison, where the isolation of rat cardiac cells was performed according to a previously report (14). Briefly, ventricles were manually minced with a surgical knife and incubated in Hanks’ balanced salts solution containing collagenase in an Erlenmeyer flask that was put in a shaking water-bath at 37 C for 50 min. Static culture was performed for 10 min, and then shaking culture (130 rpm) was performed for 40 min. During the shaking culture, the collagenase solution was exchanged with new collagenase solution every 10 min. After being filtered through a membrane filter (pore size: 40 mm), the cell suspensions were put in a 50 mL tube containing an equal volume of Hanks’ balanced salts solution with 50% FBS and centrifuged (1700 g for 5 min at 4 C). The collected cells were suspended in the same culture medium described above. The isolated cells were counted by a counting chamber after treatment with a DNA-binding dye, Hoechst 33342 (Invitrogen). Only nuclear positive cells were counted to avoid contamination with erythrocytes. Microscopic photographs of cardiac cells were observed by a phase-contract microscope (ET300, Nikon, Tokyo), and the images were recorded by a digital video camera (DCR-TRV900, Sony, Tokyo) through a CCD camera (HV-D28S, Nikon). Isolated cells were plated onto a 4-well culture dish at a concentration of 6.0 105 cells/well. After being cultured for 4 days, the cultured cells were fixed with 4% paraformaldehyde and permeabilized with 0.15% Triton X-100 in phosphate-buffered saline; after which, indirect immunofluorescence assays were performed. Cardiac troponin T antibody (mouse monoclonal antibody, Funakoshi, Tokyo) and Alexa-Fluor 488-labeled anti-mouse IgG antibody (Invitrogen) were used as the primary and secondary antibodies, respectively. Prepared specimens were examined by a fluorescence microscope (ELIPSE TE2000-U, Nikon) with a CCD camera (Axio Cam HRc, Carl Zeiss, Hallbergmoos, Germany). Creatine kinase (CK) is an enzyme normally found in cardiomyocytes so that CK activity can be used as an index for estimating the relative ratio of cardiomyocytes per isolated cells. After being centrifuged, the collected cells (6.0 105 cells) were lysed with 0.15% Triton X-100, and CK activity was measured by an enzymatic method at an outsourcing laboratory, SRL (Tokyo). CK activity was measured per 6.0 105 of isolated cells. Normally existing in the soluble fractions of cells, when CK is found in blood it indicates cell injury (14,15). Therefore, CK activity measurement can indicate the viability of cardiomyocytes after cell isolation. After 4 days of cultivation, the CK activity in both the cells and in the culture supernatants was measured. The CK activity in the cardiomyocytes was measured after treatment with 0.15% Triton X-100. The viability of cardiomyocytes was calculated by the following equation;
Relative CK activityð%Þ ¼
CKact-cell 100 CKact-cell þ CKact-medium
(1)
CKact-cell and CKact-medium are CK activities in the cells and in the culture supernatants, respectively. In addition, cell viability was also measured by the LIVE/DEAD Viability/Cytotoxicity Kit (Invitrogen), which contains calcein AM and ethidium homodimer-1. Calcein AM, which is a membrane-permeant reagent, is cleaved by esterases in live cells to yield cytoplasmic green fluorescent dye, and membrane-impermeant ethidium homodimer-1 is inserted into the nucleic acids of membrane-injured cells with red fluorescence. Briefly, isolated cardiac cells were cultured for 4 days and cells were stained with calcein AM and ethidium homodimer-1 for 30 min at 37 C. The fluorescent images of the cells were photographed by a fluorescence microscope (ECLIPSE TE2000-U) with a CCD camera (Axio Cam HRc, Carl Zeiss). The number of isolated cells per weight of heart tissue, CK activity per unit number of isolated cells, and the viability of cardiomyocytes that were isolated by both methods were examined. The mean and standard deviation of the data from each individual technician were obtained, and an unpaired Student’s t-test was performed to compare both methods. Experimental errors of the new system and the conventional method were estimated by the coefficient of variation (CV). CV was calculated by the following equation: CV ¼
Standard deviations 100 Means
(2)
A p-value of less than 0.01 was considered significant. The isolated cardiac cells were plated onto a 35 mm diameter temperature-responsive culture dish (UpcellⓇ) (CellSeed, Tokyo) at a concentration of 2.4 106 cells/dish. After 4 days, to release the confluent cells as an intact sheet, the culture dishes were placed in a CO2 incubator set at 20 C. The obtained cell sheet was fixed with 4% paraformaldehyde and embedded in paraffin, sectioned, and stained with hematoxylin and eosin. Prepared specimens were examined by a microscope (ELIPSE E800, Nikon). In the new method, a rotation speed of 200 rpm for 30 min with an interruption (0.5-s rotation and 1-s interruption) (Movie S1) was used for the following reasons. (i) The ratio of cardiac troponin Tpositive cells after incubation for 4 days decreased by increasing the rotation speed from 200 rpm to 500 rpm (Fig. 2AeD). (ii) Similar results were obtained in CK activity for a unit number of isolated cells (data not shown). In addition, cardiac cells isolated with a rotation speed of 500 rpm beat only partly, while cardiac cells isolated with a rotation speed of 200 rpm beat completely and synchronously (Movies S2 and S3). (iii) When a rotation speed of 100 rpm was used, the number of isolated cells was less than at
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FIG. 2. Immunocytochemistry of cardiac cells in the isolated cells. The cells were isolated for 15 min (AeD) and for 30 min (E). Rotation speed: (A, E) 200 rpm; (B) 300 rpm; (C) 400 rpm; and (D) 500 rpm. In the conventional manual method cells were incubated for 50 min (F). After isolated cells were counted, these cells were plated onto a 4-well culture dish at a concentration of 6.0 105 cells/well. Cardiac troponin T in the cultured cardiomyocytes was stained green with a cardiac-specific troponin T antibody. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
200 rpm (data not shown). (iv) The number of isolated cells increased time-dependently up to 30 min (data not shown). The number of isolated cells using interrupted rotation was greater than with continuous rotation (data not shown). The numbers of isolated cells per weight of heart tissue by the new method (rotation of 200 rpm for 30 min) and the conventional method (incubation for 50 min) were (9.7 1.8) 104 cells/mg and (5.9 1.3) 104 cells/ mg, respectively (Fig. 3A). The difference between the results was statistically significant. CK activities per unit number of isolated cells by the new method and the conventional method were 488.4 25.0 U/1.2 106 cells and 478.4 108.5 U/1.2 106 cells, respectively (Fig. 3B). The viabilities of cardiomyocytes isolated by the new method and the conventional method were 98.5 0.3% and 95.5 1.3%, respectively (Fig. 3C). Similar results were shown by LIVE/DEAD assay, namely, cells isolated in both cell isolation systems largely lived (Fig. S1). Here again the difference between the two methods was statistically significant. These results showed that the number of isolated cells and the viability of cardiomyocytes improved using the new method, while the relative ratio of cardiomyocytes was almost equal. The number of cardiac troponin T-positive cells after incubation for 4 days isolated at 200 rpm for 30 min were almost equal to that of the conventional method (Fig. 2E and F). In addition, the CV of the number of isolated cells, the CK activity, and the viability of cardiomyocytes from the new system were less than those of the conventional method. In
J. BIOSCI. BIOENG., particular the CV of CK activity, and the viability of cardiac cells with the new system were extremely low (Table S1). These results show that the reproducibility of data from experiments by the new method was higher than with the conventional method. This supports the premise that the new semi-automatic system reduces individual variation of results in the isolation of living cells, which in turn will produce better results in tissue-engineering applications that rely on a consistent source of living cells. To confirm the actual beating function of cardiomyocytes, the isolated cells were carefully observed. Spontaneous beating was observed in isolated cardiac cells at least 1 day after the seeding onto culture dishes, and these cells then gradually developed synchronous beating (Movie S2). These results show that functional and living cardiomyocytes could be isolated by the new method just like the conventional method. There are several reports regarding methods for cardiac cell isolation (6,16), and here we present a number of significant improvements in a new cell isolation system. Cardiomyocytes having the potential to beat were isolated from neonatal rat hearts by using this new isolation system. In this study, the optimal conditions were examined in detail; namely, optimal rotation speed, condition, and the duration of rotation. The optimal conditions increased the efficiency of cell isolation. Consequently, the new system was able to isolate approximately 1.6 times more cells than the conventional manual method, while the isolation time of the new method was almost half that of the conventional method (Fig. 3). The new system also increased the viability of cardiomyocytes (Fig. 3). Therefore, the new isolation system is thought to provide a significant improvement in cell isolation for use in regenerative medicine. In our cell isolation system, 200 rpm was the optimal speed for isolating beating cardiomyocytes from heart tissues. On the other hand, to isolate cells from other tissues, the optimal conditions of the new isolation system could vary depending on target cell type. Recently, several reports about automatic cell culture systems have been published (17,18). Automation that removes manual manipulation has been advanced in the field of tissue-engineering and regenerative medicine, because manual manipulation increases the possibility of human error, including infectious contamination. In this report, the mincing of tissues/organs and cell isolation with enzymes were automated to avoid possible adverse effects due to variations of individual technicians which could affect the viabilities, conditions, and proportions of the isolated cells. In fact, the CVs of all items in the conventional manual method were larger than those in the new method (Table S1). Although, the difference in the CVs of the relative ratio of cardiac cells and the relative CK activity between the two methods was large, the difference in the CV of the number of isolated cells was small (Table S1). Variation in cell density of individual tissues, which is beyond the control of our isolation system, might induce variation in the number of isolated cells. Constancy in the CVs of the relative ratio of cardiac cells and the viabilities of cardiac cells should contribute to more consistent quality of isolated cells, but not the number of isolated cells. Consistency of cell quality is important for tissue-engineering and regenerative medicine, which makes our isolation system an important improvement in the development of tissue-engineering and regenerative medicine. At present our system requires pretreatment to isolate homogenous tissue before cell isolation. In the case of complex tissue, additional pretreatment is necessary before cell isolation. For example, in the isolation of adipose tissue-derived stem cells, small vessels and fibrous tissue should first be removed from the adipose tissue. Further developments of a cell isolation system might require the addition of robot technology, for example, to establish a truly automated cell isolation system that includes the isolation of homogenous tissue. In the case of using a temperature-responsive culture dish, when culture temperature was decreased from 37 C to 20 C, cardiac cells detached themselves as a contiguous cell sheet within 1 h. The
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FIG. 3. Effect of the new system and the conventional method on the number of isolated cells per weight of heart tissue, the relative ratio of cardiomyocytes, and the relative creatine kinase activity. Three persons independently performed each experiment. A rotation of 200 rpm for 30 min with an interruption rotation (0.5-s rotation and 1-s interruption) was used in the new method. The number of isolated cells per weight of heart tissue (A), the relative CK activity per isolated cells (B) and the relative CK activity (C) were measured as followed: Nuclear positive cell numbers ðcellsÞ The number of isolated cells per weight of the heart tissues ¼ Total weight of heart tissues ðmgÞ Creatine kinase activity ðUÞ The relative ratio of cardiomyocytes per the isolated cells ¼ 1:2 106 nuclear positive cells CKact-cell The relative CK activityð%Þ ¼ 100 CKact-cell þ CKact-medium where CKact-cell and CKact-medium are CK activities in the cells and CK activities in the culture supernatants, respectively. The data are expressed as mean standard deviation (**p < 0.01, n ¼ 5).
released cardiac cell sheets shrank due to cytoskeletal tensile reorganization (Fig. S2A) (the area of sheet: approximately 9.6 cm2 / 2.4 cm2), resulting in a double- or triple-layered cell sheet. The thickness of the cell sheet was found to be approximately 20e30 mm (Fig. S2B). The harvested cell sheet continued to show a synchronous and spontaneous beating even after detachment (Movie S4). These results showed that an electrical communicative and functional cardiac cell sheet could be fabricated from cardiac cells isolated by the new method, just like the conventional method. In conclusion, a new-automated system was developed to isolate living cells from tissue/organs and was demonstrated by isolating beating cardiac cells from heart tissue. Our new system isolated higher quality cells and reduced cell isolation time while also improving the viability of the cells. All current and future applications in regenerative medicine are dependent on a consistent and reliable supply of high quality, viable living cells. We are confident that this new isolation system can be a powerful new tool for use in the fields of bioengineering, tissue-engineering and regenerative medicine. Supplementary data related to this article can be found online at http://dx.doi.org/10.1016/j.jbiosc.2012.08.013. We appreciate the useful comments and technical criticism from Prof. Hiroshi Iseki and Dr. Norio Ueno (Institute of Advanced Biomedical Engineering and Science, Tokyo Women’s Medical University). As well as the support and encouragement of Prof. Sunao Takeda (He was with Nihon Kohden and is now with the Dept. of Clinical Engineering, Tokyo Univ. of Technology, Tokyo, Japan), Shinji Yamamori, Hirotsugu Kubo and Akane Suzuki (Nihon Kohden). This work is granted by the Japan Society for the Promotion of Science (JSPS) through the “Funding Program for WorldLeading Innovative R&D on Science and Technology (FIRST Program),” initiated by the Council for Science and Technology Policy (CSTP) and is supported by grants from the Global Center of Excellence (GCOE) Program, Multidisciplinary Education and Technology and Research Center for Regenerative Medicine (MERCREM) from the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan.
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