Original Article

Proficiency of Carboxymethyl cellulose as a Cryoprotectant. Clinical and Histological Evaluation of Cryopreserved Heterogenous Mesenchymal Stem Cell-Exosomal Hydrogel on Critical Size Skin Wounds in Dogs


Background: Fresh stem cell exosomes are usually obtained and reused in the same individual. It cannot be kept viable for a long period of time regardless of the lengthy preparation time. Freezing is typically used to preserve the viability of perishable materials and increase their lifetime. Regrettably, normal freezing of biomaterials leads to cell damage. Therefore, a cryoprotectant can save the cells from the conventional cryodamage. Sodium carboxymethylcellulose (NA-CMC) is a powdery substance that is used to manufacture bio-safe hydrofilm gels because of its high viscosity, cytocompatibility, and non-allergenic nature.

Materials and Methods: Sterile CMC hydrogel was prepared, part of which was loaded with exosomal solution derived from MSCs. The gel was kept at −20°C for preservation. Two bilateral full-thickness circular skin wounds of 2-cm diameter were created on the back of experimental dogs. The wounds were at least 2.5 cm apart. Treatment started 24 hours after wound creation. Group I received CMC gel solely, whereas group II received frozen CMC exosomal gel. The gel was applied 4 times, a single application per day with 1-day interval.

Results: Clinically, the frozen exosomal gel significantly promoted wound healing with no scaring. Histologically, enhanced dermal fibroblasts and organized collagen deposition were seen in the treated group.

Conclusion: CMC proved to be an efficient cryoprotectant and a suitable vehicle for exosomes. Deep freezing was proven to conserve the viability, extended the preservation, and facilitated the usage of exosomal gel. This technique of preserved cell-free therapy is inexpensive, time-saving, and proficient and seems suitable for treating cutaneous wounds.

1. Lötvall J, Hill AF, Hochberg F, et al. Minimal experimental requirements for definition of extracellular vesicles and their functions: a position statement from the International Society for Extracellular Vesicles. J Extracell Vesicles 2014;3:26913.
2. Camussi G, Deregibus MC, Cantaluppi V. Role of stem-cell-derived microvesicles in the paracrine action of stem cells. Biochem Soc Trans . 2013;41(1):283-7.
3. del Conde I, Shrimpton CN, Thiagarajan P, et al. Tissue-factor–bearing microvesicles arise from lipid rafts and fuse with activated platelets to initiate coagulation. Blood. 2005;106(5):1604–1611.
4. Bruno S, Grange C, Collino F, et al. Microvesicles derived from mesenchymal stem cells enhance survival in a lethal model of acute kidney injury. PLoS One. 2012;7(3):e33115.
5. Sudhakar Y, Kuotsu K, Bandyopadhyay AK. Buccal bioadhesive drug delivery—a promising option for orally less efficient drugs. J Control release. 2006;114(1):15–40.
6. Castellano JJ, Shafii SM, Ko F, et al. Comparative evaluation of silver‐containing antimicrobial dressings and drugs. Int Wound J. 2007;4(2):114–122.
7. Capanema NS V, Mansur AAP, de Jesus AC, et al. Superabsorbent crosslinked carboxymethyl cellulose-PEG hydrogels for potential wound dressing applications. Int J Biol Macromol. 2018;106:1218–1234.
8. Shimamura MK, Kamata K, Yao A, et al. Scattering functions of knotted ring polymers. Phys Rev E Stat Nonlin Soft Matter Phys . 2005;72(4 Pt 1):041804.
9. El-Tookhy OS, Shamaa AA, Shehab GG, et al. Histological evaluation of experimentally induced critical size defect skin wounds using exosomal solution of mesenchymal stem cells derived microvesicles. Int J stem cells.; 2017;10(2):144-153.
10. Jung D-I, Ha J, Kim J-W, et al. Canine mesenchymal stem cells derived from bone marrow: isolation, characterization, multidifferentiation, and neurotrophic factor expression in vitro. J Vet Clin. 2008;25(6):458–465.
11. Ryu H-H, Kang B-J, Park S-S, et al. Comparison of mesenchymal stem cells derived from fat, bone marrow, Wharton’s jelly, and umbilical cord blood for treating spinal cord injuries in dogs. J Vet Med Sci. 2012; 74(12):1617-30.
12. Théry C, Amigorena S, Raposo G, et al. Isolation and characterization of exosomes from cell culture supernatants and biological fluids. Curr Protoc cell Biol. 2006 Apr;Chapter 3:Unit 3.22.
13. Adam JA. A simplified model of wound healing (with particular reference to the critical size defect). Math Comput Model. 1999;30(5–6):23–32.
14. Karayannopoulou M, Tsioli V, Loukopoulos P, et al. Evaluation of the effectiveness of an ointment based on Alkannins/Shikonins on second intention wound healing in the dog. Can J Vet Res. 2011;75(1):42–48.
15. Kim J, Lee J, Lyoo YS, et al. The effects of topical mesenchymal stem cell transplantation in canine experimental cutaneous wounds. Vet Dermatol. 2013;24(2):242-e53.
16. Zhang J, Guan J, Niu X, et al. Exosomes released from human induced pluripotent stem cells-derived MSCs facilitate cutaneous wound healing by promoting collagen synthesis and angiogenesis. J Transl Med. 2015;13:49.
17. Rani S, Ryan AE, Griffin MD, et al. Mesenchymal stem cell-derived extracellular vesicles: toward cell-free therapeutic applications. Mol Ther. 2015;23(5):812–823.
18. Togel F, Weiss K, Yang Y, et al. Vasculotropic, paracrine actions of infused mesenchymal stem cells are important to the recovery from acute kidney injury. Am J Physiol Physiol. 2007;292(5):F1626–35.
19. Simons M, Raposo G. Exosomes–vesicular carriers for intercellular communication. Curr Opin Cell Biol. 2009;21(4):575–81.
20. Kinnaird T, Stabile E, Burnett MS, et al. Marrow-derived stromal cells express genes encoding a broad spectrum of arteriogenic cytokines and promote in vitro and in vivo arteriogenesis through paracrine mechanisms. Circ Res. 2004;94(5):678–85.
21. Yamashita T, Takahashi Y, Takakura Y. Possibility of exosome-based therapeutics and challenges in production of exosomes eligible for therapeutic application. Biol Pharm Bull. 2018;41(6):835–842.
22. Li X, Corbett AL, Taatizadeh E, et al. Challenges and opportunities in exosome research—Perspectives from biology, engineering, and cancer therapy. APL Bioeng. 2019;3(1):011503.
23. Ha D, Yang N, Nadithe V. Exosomes as therapeutic drug carriers and delivery vehicles across biological membranes: current perspectives and future challenges. Acta Pharm Sin B. 2016;6(4):287–96.
24. Lou G, Chen Z, Zheng M, et al. Mesenchymal stem cell-derived exosomes as a new therapeutic strategy for liver diseases. Exp Mol Med. 2017;49(6):e346.
25. Mendt M, Kamerkar S, Sugimoto H, et al. Generation and testing of clinical-grade exosomes for pancreatic cancer. JCI Insight. 2018; 3(8):e99263.
26. Nakamura K, Sawada K, Kinose Y, et al. Exosomes promote ovarian cancer cell invasion through transfer of CD44 to peritoneal mesothelial cells. Mol Cancer Res. 2017;15(1):78–92.
27. He M, Qin H, Poon TCW, et al. Hepatocellular carcinoma-derived exosomes promote motility of immortalized hepatocyte through transfer of oncogenic proteins and RNAs. Carcinogenesis. 2015;36(9):1008–18.
28. Pollock K, Stroemer P, Patel S, et al. A conditionally immortal clonal stem cell line from human cortical neuroepithelium for the treatment of ischemic stroke. Exp Neurol. 2006;199(1):143–55.
29. Chen TS, Arslan F, Yin Y, et al. Enabling a robust scalable manufacturing process for therapeutic exosomes through oncogenic immortalization of human ESC-derived MSCs. J Transl Med. 2011;9:47.
30. Zhu X, Badawi M, Pomeroy S, et al. Comprehensive toxicity and immunogenicity studies reveal minimal effects in mice following sustained dosing of extracellular vesicles derived from HEK293T cells. J Extracell Vesicles. 2017;6(1):1324730.
31. Abd‐Allah SH, El‐Shal AS, Shalaby SM, et al. The role of placenta‐derived mesenchymal stem cells in healing of induced full‐thickness skin wound in a mouse model. IUBMB Life. 2015;67(9):701–9.
32. Basiouny HS, Salama NM, El Maadawi ZM, et al. Effect of bone marrow derived mesenchymal stem cells on healing of induced full-thickness skin wounds in albino rat. Int J Stem Cells. 2013;6(1):12-25.
33. Tark K-C, Hong J-W, Kim Y-S, et al. Effects of human cord blood mesenchymal stem cells on cutaneous wound healing in leprdb mice. Ann Plast Surg. 2010;65(6):565–72.
34. Falanga V, Iwamoto S, Chartier M, et al. Autologous bone marrow–derived cultured mesenchymal stem cells delivered in a fibrin spray accelerate healing in murine and human cutaneous wounds. Tissue Eng. 2007;13(6):1299–312.
35. Petrilli R, Lopez RFV. Brasil- methods for topical skin drug delivery: concepts and applications. Braz J Pharm. Sci. 2018; 54 (spe).
36. Lee M, Ban J-J, Im W, et al. Influence of storage condition on exosome recovery.. Bioprocess Biosyst Eng. 2016;21(2):299–304.
37. Kusuma GD, Barabadi M, Tan JL, et al. To protect and to preserve: novel preservation strategies for extracellular vesicles. Front Pharmacol. 2018;9:1199.
38. Bennett TP, Frieden E. Worthington publication which was originally published in 1972 as the Manual of Clinical Enzyme Measurements: Modern Topics in Biochemistry. Macmillan, London; 1969.
39. Janz F de L, Debes A de A, Cavaglieri R de C, et al. Evaluation of distinct freezing methods and cryoprotectants for human amniotic fluid stem cells cryopreservation. Biomed Res Int. 2012;2012;1-10.
40. Witwer KW, Buzas EI, Bemis LT, et al. Standardization of sample collection, isolation and analysis methods in extracellular vesicle research. J Extracell Vesicles. 2013; 2.
41. Zhou H, Yuen PST, Pisitkun T, et al. Collection, storage, preservation, and normalization of human urinary exosomes for biomarker discovery. Kidney Int. 2006;69(8):1471–6.
42. Munagala R, Aqil F, Jeyabalan J, et al. Bovine milk-derived exosomes for drug delivery. Cancer Lett. 2016;371(1):48–61.
43. Sokolova V, Ludwig A-K, Hornung S, et al. Characterisation of exosomes derived from human cells by nanoparticle tracking analysis and scanning electron microscopy. Colloids Surf B Biointerfaces . 2011; 87(1):146-50.
44. Lv LL, Cao Y, Liu D, et al. Isolation and quantification of microRNAs from urinary exosomes/microvesicles for biomarker discovery. Int J Biol Sci. 2013;9(10):1021-31.
45. Ge Q, Zhou Y, Lu J, et al. miRNA in plasma exosome is stable under different storage conditions. Molecules. 2014;19(2):1568–75.
46. Eroglu A, Russo MJ, Bieganski R, et al Intracellular trehalose improves the survival of cryopreserved mammalian cells. Nat Biotechnol. 2000;18(2):163-7.
47. Bosch S, De Beaurepaire L, Allard M, et al. Trehalose prevents aggregation of exosomes and cryodamage. Sci Rep. 2016;6:36162.
48. Ramli NA, Wong TW. Sodium carboxymethylcellulose scaffolds and their physicochemical effects on partial thickness wound healing. Int J Pharm. 2011;403(1–2):73–82.
49. Field CK, Kerstein MD. Overview of wound healing in a moist environment. Am J Surg. 1994;167(1A): 2S-6S.
50. Stashak TS, Farstvedt E, Othic A. Update on wound dressings: indications and best use. Clin Tech Equine Pract. 2004;3(2):148–163.
51. Rodrigues C, de Assis AM, Moura DJ, et al. New therapy of skin repair combining adipose-derived mesenchymal stem cells with sodium carboxymethylcellulose scaffold in a pre-clinical rat model. PLoS One. 2014;9(5):e96241.
52. Wu M, Ouyang Y, Wang Z, et al. Isolation of exosomes from whole blood by integrating acoustics and microfluidics. Proc Natl Acad Sci U S A . 2017 Oct 3;114(40):10584-10589.
53. Yuana Y, Levels J, Grootemaat A, et al. Co-isolation of extracellular vesicles and high-density lipoproteins using density gradient ultracentrifugation. J Extracell Vesicles. 2014;3.
54. Iacono E, Lanci A, Merlo B, et al. Effects of amniotic fluid mesenchymal stem cells in carboxymethyl cellulosegel on healing of spontaneous pressure sores: clinical outcome in sevenhospitalized neonatal foals. Turkish J Biol. 2016;40(2):484–492.
55. Fu X, Fang L, Li X, et al. Enhanced wound‐healing quality with bone marrow mesenchymal stem cells autografting after skin injury. Wound Repair Regen. 2006;14(3):325-35.
IssueVol 15, No 3 (2021) QRcode
SectionOriginal Article(s)
DOI https://doi.org/10.18502/ijhoscr.v15i3.6848
Critical-size defect; Skin wound; Heterogeneous; Frozen; Exosome; Carboxymethyl cellulose

Rights and permissions
Creative Commons License This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License.
How to Cite
Bahr M, Amer M, Abo-EL-Sooud K, Abdallah A, Shehab G, El-Tookhy O. Proficiency of Carboxymethyl cellulose as a Cryoprotectant. Clinical and Histological Evaluation of Cryopreserved Heterogenous Mesenchymal Stem Cell-Exosomal Hydrogel on Critical Size Skin Wounds in Dogs. Int J Hematol Oncol Stem Cell Res. 2021;15(3):178-191.