Mehmet Ali KARACA, Derya DİLEK KANÇAĞI, Uğur ÖZBEK, Ercüment OVALI, Ozgul GOK

Mezankimal Kök Hücre Enkapsülasyonu için 3B Baskı ile Makro Kapsül Üretimi

Amaç: Hücre nakli, vücutta hücre aracılı bağışıklık reaksiyonlarını indüklemek için yaygın olarak kullanılan bir yöntemdir. Bununla birlikte, nakledilen hücrelere karşı olası bağışıklık tepkileri, uygulanan hücre tedavisinin etkinliğini azaltır. Bu sorun, hücreleri enkapsüle eden ve onları bağışıklık sistemi saldırılarından koruyan 3D baskılı polimerik kapsüller aracılığıyla hücrelerin nakli ile çözülebilir. Hücre yüklü kapsüller (makro veya mikro), daha etkili hücresel tedaviler için potansiyel taşıyıcılar olarak ortaya çıkmıştır. Bu çalışmada, biyobozunur ve biyouyumlu polyester “polikaprolakton (PCL)”den 3D baskılı gözenekli kapsüller hazırlanmış ve bu yarı-geçirgen makro-kapsüllerin hücre transplantasyonu için etkin bir taşıyıcı olup olmadığı incelenmiştir. Yöntem: Tasarlanan makro kapsül (2*5*10 mm) Autodesk Fusion 360 programında çizilmiş ve polikaprolakton (PCL) materyali ile Axo Bioprinter Dual Print Head System kullanılarak basılmıştır. Makro-kapsülün kapalı formu üzerinde sızıntı, yüzey elektron mikroskobu ile görsel olarak kontrol edilmiştir. Makrokapsülün geçirgenliği tripan mavi boya ve ayrıca insan serum albümin (HSA) proteini ile test edilmiştir. Polikaprolakton malzemelerinin hücreler üzerindeki sitotoksisitesi 24 ve 72 saatlik zaman dilimlerinde test edilmiştir. Bulgular: Elde edilen sonuçlara göre kapalı ve içi boş olacak şekilde yarı-geçirgen makro-kapsül formu başarı ile elde edildi. Makro-kapsülün gözenekli yapısının olduğu kapsülün tripan mavi boya kullanılarak gösterildi. Makrokapsülün gözenekliliğinin değerlendirilmesi için makro-kapsülden insan serum albümin protein salınımı yapıldı. İnsan serum albümin (HSA) proteininin miktarca %98'inin 24 saat içinde makro kapsülden dışarıya salındığı gösterildi. Polikaprolakton (PCL) makro-kapsül UV ışığı kullanılarak sterilize edildi ve in vitro şartlarda sitotoksisite göstermediği raporlandı. Ayrıca makro-kapsül içindeki hücrelerin 12 günlük inkübasyon sırasında 2D hücre kültürü şartlarına göre en az %10 oranında dışarıdaki besiyerden glikoz tüketimi yaptıkları ve ürettikleri laktik asit moleküllerinin en az %8’ini içerden dışarıya salabildikleri gösterildi. Sonuç: Polimer makro-kapsülün tekrarlanabilir üretimi, içerideki enkapsüle hücrelerin yüksek canlılık oranı ve bunların metabolik değerlendirme sonuçları, bu kapsüllerin transplantasyona bağlı hücresel terapilerde canlı hücreler için etkin taşıyıcılar olma potansiyelini açıkça göstermiştir.

Anahtar Kelimeler:

Makro Enkapsülasyon, Hücresel Tedavi, Enkapsülasyon, polimerik kapsül, 3B Basım, mezenkimal kök hücre

Macro-Capsule Fabrication via 3D Printing for Mesenchymal Stem Cell Encapsulation

Purpose: Cell transplantation is a widely used method to induce cell-mediated immune reactions inside the body. However, possible immune responses to the transplanted cells decrease the efficiency of applied cell therapy. This issue can be addressed by the transplantation of cells via 3D-printed polymeric capsules which encapsulate cells and protect them from immune system attacks. Cell-loaded capsules (macro or micro) have emerged as potential carriers for more efficacious cellular therapies. In this study, 3D-printed porous capsules were prepared from biodegradable and biocompatible polyester “polycaprolactone (PCL)” and this macro-capsule was evaluated as a carrier for its cell encapsulation effectiveness. Method: The macro-capsule was designed to have dimensions of 2x5x10 mm and drawn in Autodesk Fusion 360 program. PCL was utilized for its 3D bio-printing via Axolotl Bioprinter Dual Print Head System. Leakage on the closed form of the macro-capsule was visually controlled by surface electron microscopy (SEM). Permeability of the macro-capsule was tested with trypan blue dye and human serum albumin (HSA) protein. Sterilization of the obtained macro-capsule was achieved via UV light and the cytotoxicity of the polycaprolactone capsule was tested for 24 and 72 hour incubation time periods. Results: The semi-permeable macro-capsule was successfully obtained as closed and hollow form. Its porous structure was demonstrated using trypan blue dye. To evaluate the porosity of the macrocapsule, human serum albumin (HSA) protein release was performed from the macrocapsule. It has been shown that 98% of HSA was released from the macrocapsule within 24 hours. The polycaprolactone (PCL) macrocapsule was sterilized using UV light and was reported to show no in vitro cytotoxicity. In addition, it was shown that the cells in the macro-capsule consumed at least 10% glucose from the outside medium during 12 days of incubation, compared to 2D cell culture conditions, and were able to release at least 8% of the lactic acid molecules outside. Conclusion: In conclusion, reproducible fabrication of polymer macro-capsule, high viability of encapsulated cells inside, and their metabolic assessment results have obviously indicated the potential of these capsules as effective carriers for living cells with transplantation-dependent cellular therapies.

Keywords:

Macro-capsule, Polymeric Capsule, 3D printing, Cell therapy, Mesenchymal Stem Cells,

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Moshaverinia A, Xu X, Chen C, et al. Application of stem cells derived from the periodontal ligament orgingival tissue sources for tendon tissue regeneration. Biomaterials. 2014;35(9). doi:10.1016/j.biomaterials.2013.12.053
Kook YM, Kang YM, Moon SH, Koh WG. Bi-compartmental 3D scaffolds for the co-culture of intervertebral disk cells and mesenchymal stem cells. Journal of Industrial and Engineering Chemistry. 2016;38. doi:10.1016/j.jiec.2016.04.013
Huebsch N, Lippens E, Lee K, et al. Matrix elasticity of void-forming hydrogels controls transplanted-stem-cell-mediated bone formation. Nature Materials. 2015;14(12). doi:10.1038/nmat4407
Park JS, Shim MS, Shim SH, et al. Chondrogenic potential of stem cells derived from amniotic fluid, adipose tissue, or bone marrow encapsulated in fibrin gels containing TGF-β3. Biomaterials. 2011;32(32). doi:10.1016/j.biomaterials.2011.07.043
Krishnan R, Alexander M, Robles L, Foster CE, Lakey JRT. Islet and stem cell encapsulation for clinical transplantation. Review of Diabetic Studies. 2014;11(1):84-101. doi:10.1900/RDS.2014.11.84
Desai TA, Tang Q. Islet encapsulation therapy — racing towards the finish line? Nature Reviews Endocrinology. 2018;14(11):630-632. doi:10.1038/s41574-018-0100-7
Saenz Del Burgo L, Ciriza J, Espona-Noguera A, et al. 3D Printed porous polyamide macrocapsule combined with alginate microcapsules for safer cell-based therapies. Scientific Reports. 2018;8(1):1-14. doi:10.1038/s41598-018-26869-5
O’sullivan ES, Vegas A, Anderson DG, Weir GC. Islets Transplanted in Immunoisolation Devices: A Review of the Progress and the Challenges that Remain. Published online 2011. doi:10.1210/er.2010-0026
An D, Chiu A, Flanders JA, et al. Designing a retrievable and scalable cell encapsulation device for potential treatment of type 1 diabetes. doi:10.1073/pnas.1708806115
Evron Y, Colton CK, Ludwig B, et al. Long-term viability and function of transplanted islets macroencapsulated at high density are achieved by enhanced oxygen supply OPEN. 2018;8:6508. doi:10.1038/s41598-018-23862-w
Weir GC, Bonner-Weir S. Perspectives in Diabetes Scientific and Political Impediments to Successful Islet Transplantation. Vol 46.; 1997.
Pipeleers D, Ziekenhuis Brussel U, Gillard BP, Strand BL. (No Title). 2013;56:1605-1614. doi:10.1007/s00125-013-2906-0
Scharp DW, Marchetti P. Encapsulated islets for diabetes therapy: History, current progress, and critical issues requiring solution. Advanced Drug Delivery Reviews. 2014;67-68:35-73. doi:10.1016/j.addr.2013.07.018
Gabr MM, Zakaria MM, Refaie AF, et al. Insulin-producing Cells from Adult Human Bone Marrow Mesenchymal Stromal Cells Could Control Chemically Induced Diabetes in Dogs: A Preliminary Study. doi:10.1177/0963689718759913
Skrzypek K, Groot Nibbelink M, Van Lente J, et al. Pancreatic islet macroencapsulation using microwell porous membranes OPEN. doi:10.1038/s41598-017-09647-7
Tomei AA, Villa C, Ricordi C. Development of an encapsulated stem cell-based therapy for diabetes. Expert Opinion on Biological Therapy. 2015;15(9):1321-1336. doi:10.1517/14712598.2015.1055242
Vaithilingam V, Evans MDM, Lewy DM, Bean PA, Bal S, Tuch BE. Australian Foundation for Diabetes Research. New South Wales. (2). doi:10.1038/s41598-017-10359-1
Fobker M. Stability of glucose in plasma with different anticoagulants. Clinical Chemistry and Laboratory Medicine. 2014;52(7):1057-1060. doi:10.1515/cclm-2013-1049
Ryan EA, Paty BW, Senior PA, et al. Five-year follow-up after clinical islet transplantation. Diabetes. 2005;54(7). doi:10.2337/diabetes.54.7.2060
Kumar PV, Jain NK. Suppression of Agglomeration of Ciprofloxacin-Loaded Human Serum Albumin Nanoparticles.; 2007.
Giustarini D, Dalle-Donne I, Milzani A, Rossi R. Low molecular mass thiols, disulfides and protein mixed disulfides in rat tissues: Influence of sample manipulation, oxidative stress and ageing. Mechanisms of Ageing and Development. 2011;132(4):141-148. doi:10.1016/j.mad.2011.02.001
Olabisi RM. The Authors. Journal of Biomedical Materials Research Part A. 2014;103:846-859. doi:10.1002/jbm.a.35205
Maitz MF. Applications of synthetic polymers in clinical medicine. Biosurface and Biotribology. 2015;1(3):161-176. doi:10.1016/j.bsbt.2015.08.002
Rivera Diaz PA, Gómez Camargo DE, Ondo-Méndez A, Gómez-Alegría CJ. A colorimetric bioassay for quantitation of both basal and insulin-induced glucose consumption in 3T3-L1 adipose cells. Heliyon. 2020;6(2). doi:10.1016/j.heliyon.2020.e03422