Holding Solutions, Conditions, and Additives to Optimize Hair Follicle Viability and Function

  • William D. Ehringer
  • Kristyn H. Smith


Hair restoration surgery (HRS) has made great progress in regards to outcome and proper hairline design. The result is that the majority of HRS cases are considered “successful,” and this is reflected in the ever-increasing numbers of patients seeking HRS. However, issues with inconsistent growth and survival of follicular grafts have continued to plague surgeons. It is the inconsistency in patient outcomes that has focused attention on how to maintain maximal follicular graft survival. Follicular grafts are exposed to physical and biochemical trauma from the point of isolation to implantation. To address these issues, improved graft storage solutions and methods in which the grafts are isolated have been developed. Unlike organ preservation, follicular graft preservation has been poorly investigated, and there is no specific graft holding solution for HRS. In the majority of HRS, graft holding solutions are being used that are not intended for survival of follicular grafts. Further, the use of additives in the HRS process has not been sufficiently studied to understand how they may affect outcome. The purpose of this chapter is to explore the practical clinical limitations and benefits of current graft holding solutions and additives used in HRS.


Follicular unit viability Preservation Solutions Conditions Treatments 


  1. 1.
    Parsley W, Perez-Meza D. Review of factors affecting the growth and survival of follicular grafts. J Cutan Aesthet Surg. 2010;3(2):69–75. Scholar
  2. 2.
    Iwai S, Kikuchi T, Kasahara N, Teratani T, Yokoo T, Sakonju I, Okano S, Kobayashi E. Impact of normothermic preservation with extracellular type solution containing trehalose on rat kidney grafting from a cardiac death donor. PLoS One. 2012, e33157;7(3). Scholar
  3. 3.
    Guibert E, Petrenko A, Balaban C, Somov A, Rodriguez J, Fuller B. Organ preservation: current concepts and new strategies for the next decade. Transfus Med Hemother. 2011;38(2):125–42. Scholar
  4. 4.
    Philpott MP, Kealey T. Metabolic studies on isolated hair follicles: hair follicles engage in aerobic glycolysis and do not demonstrate the glucose fatty acid cycle. J Invest Dermatol. 1991;96(6):875–9.CrossRefGoogle Scholar
  5. 5.
    Crisóstomo MR, Guimarães SB, de Vasconcelos PR, Crisóstomo MG, Benevides AN. Oxidative stress in follicular units during hair transplantation surgery. Aesthetic Plast Surg. 2011;35(1):19–23. Scholar
  6. 6.
    Parsley, W. Personal communication. 2017.Google Scholar
  7. 7.
    Van Bushkirk R, Baust JM, Snyder K, Mathew A, Baust JG. Hypothermic storage and cryopreservation. BioProcess Int. 2004;2(10):42–9.Google Scholar
  8. 8.
    Rose P. Hair restoration surgery: challenges and solutions. Clin Cosmet Investig Dermatol. 2015;8:361–70. Scholar
  9. 9.
    Weinberg L, Collins N, Van Mourik K, Tan C, Bellomo R. Plasma-Lyte 148: a clinical review. World J Crit Care Med. 2016;5(4):235–50.CrossRefGoogle Scholar
  10. 10.
    Mathew A. A review of cellular biopreservation considerations during hair transplantation. Hair Transplant Forum Int. 2013;23:1.Google Scholar
  11. 11.
    Cooley J. Bio-enhanced hair restoration. Hair Transplant Forum Int. 2014;24(4):128–30.Google Scholar
  12. 12.
    Liu S, Li Z, Fu J, Sun L, Xu F, Harada T, et al. The effects of harvesting media on biological characteristics and repair potential of neural stem cells after traumatic brain injury. PLoS One. 2014;9(9):e107865. Scholar
  13. 13.
    Krueger N, Sadick N. New frontiers in graft-storage solutions. Hair Transplant. 2014;360(3):575–80.Google Scholar
  14. 14.
    Beehner ML. Notes from the editor emeritus. Hair Transplant Forum Int. 2005;15:193–5.Google Scholar
  15. 15.
    Limmer R. Micrograft survival. In: Stough D, Haber R, editors. Hair replacement. St. Louis: Mosby; 1996. p. 147–9.Google Scholar
  16. 16.
    Wise ES, Hocking KM, Eagle S, Absi T, Komalavilas P, Cheung-Flynn J, Brophy CM. Preservation solution impacts physiological function and cellular viability of human saphenous vein graft. Surgery. 2015;158(2):537–46. Scholar
  17. 17.
    Farkas J. Three myths about plasmalyte, normosol, and LR. EMCrit. 2017.Google Scholar
  18. 18.
    Ochocki J, Simon M. Nutrient-sensing pathways and metabolic regulation in stem cells. J Cell Biol. 2013;203(1):23–33. Scholar
  19. 19.
    Beehner M. 96-hour study of fu graft “out-of-body” survival comparing saline to hypothermosol/ATP solution. Hair Transplant Forum Int. 2011;21(2):33–7.Google Scholar
  20. 20.
    Meza P, Cooley J, Parsley W. Custodial vs hypothermosol. Hair graft survival at 24 and 48 hours outside the body. ISHRS Annual Meeting, San Diego, CA; 2006.Google Scholar
  21. 21.
    The effect of William E medium as a storage solution for hair transplantation. Annual meeting of International Society of Hair Restoration Surgery (ISHRS) conference, Paradise Island, Bahamas, October 23–26th, 2012.Google Scholar
  22. 22.
    Won CH, Jeong YM, Kang S, Koo TS, Park SH, Park KY, Sung YK, Sung JH. Hair-growth-promoting effect of conditioned medium of high integrin α6 and low CD 71 (α6 bri/CD71dim) positive keratinocyte cells. Int J Mol Sci. 2015;16(3):4379–91. Scholar
  23. 23.
    Yashiro M, Mii S, Aki R, Hamada Y, Arakawa N, Kawahara K, Hoffman RM, Amoh Y. From hair to heart: nestin-expressing-hair-follicle-associated pluripotent (HAP) stem cells differentiate to beating cardiac muscle cells. Cell Cycle. 2015;14(14):2362–6. Scholar
  24. 24.
    Kajiura S, Mii S, Aki R, Hamada Y, Arakawa N, Kawahara K, et al. Protocols for cryopreservation of intact hair follicle that maintain pluripotency of nestin-expressing hair-follicle-associated pluripotent (HAP) stem cells. Methods Mol Biol. 2016;1453:173–8. Scholar
  25. 25.
    Krugluger W, Moser K, Moser C, Laciak K, Hugeneck J. Enhancement of in vitro hair shaft elongation in follicles stored in buffers that prevent follicle cell apoptosis. Dermatol Surg. 2004;30(1):1–5. discussion 5PubMedGoogle Scholar
  26. 26.
    Dong L, Hao H, Xia L, Liu J, Ti D, Tong C. Treatment of MCSCs with Wnt1a-conditioned medium activates DP cells and promotes hair follicle regrowth. Sci Rep. 2014;4:5432. Scholar
  27. 27.
    Lagadic-Gossmann D, Huc L, Lecureur V. Alterations of intracellular PH homeostasis in apoptosis:origins and roles. Cell Death Differ. 2004;11(9):953–61.CrossRefGoogle Scholar
  28. 28.
    Park HJ. Effects of intracellular pH on apoptosis in HL-60 human leukemia cells. Yonsei Med J. 1995;36(6):473–9.CrossRefGoogle Scholar
  29. 29.
    Kniep EM, Roehlecke C, Ozkucur N, Steinberg A, Reber F, Knels L, Funk RH. Inhibition of apoptosis and reduction of intracellular pH decrease in retinal neural cell cultures by a blocker of carbonic anhydrase. Invest Ophthalmol Vis Sci. 2006;47(3):1185–92.CrossRefGoogle Scholar
  30. 30.
    Cassel D, Katz M, Rotman R. Depletion of cellular ATP inhibits Na+/H+ antiport in cultured human cells. J Biol Chem. 1986;261(12):5460–6.PubMedGoogle Scholar
  31. 31.
    Will MA, Clark NA, Swain JE. Biological pH buffers in IVF: help or hindrance to success. J Assist Reprod Genet. 2011;28(8):711–24. Scholar
  32. 32.
    Schaer D, Buehler P, Alayash A, Belcher JD, Vercellotti G. Hemolysis and free hemoglobin revisited: exploring hemoglobin and hemin scavengers as a novel class of therapeutic proteins. Blood. 2013;121(8):1276–84. Scholar
  33. 33.
    Dutra FF, Alves LS, Rodrigues D, Fernandez PL, de Oliveira RB, Golenbock DT, Zamboni DS, Bozza MT. Hemolysis-induced lethality involves Inflammasome activation by heme. Proc Natl Acad Sci U S A. 2014;111(39):E4110–8. Scholar
  34. 34.
    Chien S. Metabolic management. In: Madame curie bioscience database [internet]. Austin, TX: Landes Bioscience; 2000-2013.Google Scholar
  35. 35.
    Chen L, Liu C, Liu L. Changes in osmolality modulate voltage-gated calcium channels in trigeminal ganglion neurons. Brain Res. 2008;1208:56–66. Scholar
  36. 36.
    Dias C, Ala-Nissilab T, Wong-ekkabuta J, Vattulainenc I, Grant M, Karttunena M. The hydrophobic effect and its role in cold denaturation. Cryobiology. 2010;60(1):91–9. Scholar
  37. 37.
    Quinn PJ. Effects of temperature on cell membranes. Symp Soc Exp Biol. 1988;42:237–58.PubMedGoogle Scholar
  38. 38.
    Buzatu S. The temperature-induced changes in membrane potential. Riv Biol. 2009;102(2):199–217.PubMedGoogle Scholar
  39. 39.
    Angelico A, Perera M, Ravikumar R, Holroyd D, Coussios C, Mergental H, et al. Normothermic machine perfusion of decreased donor liver grafts is associated with improved postreperfusion hemodynamics. Transplant Direct. 2016;2(9):e97. Scholar
  40. 40.
    Moore A, Mercer J, Dutina G, Donahue CJ, Bauer KD, Mather JP, Etcheverry T, Ryll T. Effects of temperature shift on cell cycle, apoptosis, and nucleotide pools in CHO cell batch cultures. Cytotechnology. 1997;23(1–3):47–57. Scholar
  41. 41.
    Blair P, Flaumenhaft R. Platelet alpha-granules: basic biology and clinical correlates. Blood Rev. 2009;23(4):177–89. Scholar
  42. 42.
    Nagai M, Sato S, Kamoi H, Kamoi K. Effects of application of platelet releasate in periodontal regeneration therapy. Int J Periodontics Restorative Dent. 2005;25:571–83.  10.11607/prd.00.0664.CrossRefPubMedGoogle Scholar
  43. 43.
    Moulin V, Lawny F, Barritault D, Caruelle JP. Platelet releasate treatment improves skin healing in diabetic rats through endogenous growth factor secretion. Cell Mol Biol (Noisy-le-Grand). 1998;44(6):961–71.Google Scholar
  44. 44.
    Marx RE, Carlson ER, Eichstaedt RM, Schimmele SR, Strauss JE, Georgeff KR. Platelet-rich plasma: growth factor enhancement for bone grafts. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 1998;85(06):638–46.CrossRefGoogle Scholar
  45. 45.
    Marx RE. Platelet-rich plasma (PRP): what is PRP and what is not PRP? Implant Dent. 2004;10(04):225–8.CrossRefGoogle Scholar
  46. 46.
    Cavallo C, Roffi A, Grigolo B, et al. Platelet rich plasma: the choice of activation method affects the release of bioactive molecules. Biomed Res Int. 2016;2016:6591717. Scholar
  47. 47.
    Lubkowska A, Dolegowska B, Banfi G. Growth factor content in PRP and their applicability in medicine. J Biol Regul Homeost Agents. 2012;26(2 Suppl 1):3S–22S.PubMedGoogle Scholar
  48. 48.
    Dhurat R, Sukesh M. Principles and methods of preparation of platelet-rich plasma: a review and Author's perspective. J Cutan Aesthet Surg. 2014;7(4):189–97. Scholar
  49. 49.
    Hiraizumi Y, Fujimaki E, Transfeldt EE, Kawahara N, Fiegel VD, Knighton D, Sung JH. The effect of the platelet derived wound healing formula and the nerve growth factor on the experimentally injured spinal cord. Spinal Cord. 1996;34(7):394–402.CrossRefGoogle Scholar
  50. 50.
    Momi S, Falcinelli E, Giannini S, Ruggeri L, Cecchetti L, Corazzi T, Libert C, Gresele P. Loss of matrix metalloproteinase 2 in platelets reduces arterial thrombosis in vivo. J Exp Med. 2009;206(11):2365–79. Scholar
  51. 51.
    Sheu JR. Expression of matrix metalloproteinase-9 in human platelets: regulation of platelet activation in in vitro and in vivo studies. Br J Pharmacol. 2004;143(1):193–201.CrossRefGoogle Scholar
  52. 52.
    Renesto P, Chignard M. Tumor necrosis factor-alpha enhances platelet activation via Cathepsin G released from neutrophils. J Immunol. 1991;146(7):2305–9.PubMedGoogle Scholar
  53. 53.
    Martineau I, Lacoste E, Gagnon G. Effects of calcium and thrombin on growth factor release from platelet concentrates: kinetics and regulation of endothelial cell proliferation. Biomaterials. 2004;25(18):4489–502.CrossRefGoogle Scholar
  54. 54.
    Ho-Tin-Noé B, Goerge T, Cifuni SM, Duerschmied D, Wagner DD. Platelet granule secretion continuously prevents intratumor hemorrhage. Cancer Res. 2008;68(16):6851–8. Scholar
  55. 55.
    Olczyk P, Mencner L, Komosinska-Vassev K. The role of the extracellular matrix components in cutaneous wound healing. Biomed Res Int. 2014;2014:747584. Scholar
  56. 56.
    Huang G, Greenspan DS. ECM roles in the function of metabolic tissues. Trends Endocrinol Metab. 2012;23(1):16–22. Scholar
  57. 57.
    Lutolf M, Hubbell JA. Synthetic biomaterials as instructive extracellular microenvironments for morphogenesis in tissue engineering. Nat Biotechnol. 2005;23(1):47–55. Scholar
  58. 58.
    Gibson D, Cullen B, Legerstee R, Harding KG, Schultz G. MMPs made easy. Wounds Int. 2009;1(1):1–6.Google Scholar
  59. 59.
    Cullen B, Watt PW, Lundqvist C, Silcock D, Schmidt RJ, Bogan D, Light ND. The role of oxidised regenerated cellulose/collagen in chronic wound repair and its potential mechanism of action. Int J Biochem Cell Biol. 2002;34(12):1554–6.CrossRefGoogle Scholar
  60. 60.
    Tan K, Lawler J. The interaction of Thrombospondins with extracellular matrix proteins. J Cell Commun Signal. 2009;3(3–4):177–87. Scholar
  61. 61.
    Kivirikko KI. Collagens and their abnormalities in a wide spectrum of diseases. Ann Med. 1993;25(2):113–26.CrossRefGoogle Scholar
  62. 62.
    Yurchenco PD. Basement membranes: Cell scaffoldings and signaling platforms. Cold Spring Harb Perspect Biol. 2011;3(2):a004911. Scholar
  63. 63.
    Van der Rest M, Garrone R. Collagen family of proteins. FASEB J. 1991;5(13):2814–23.CrossRefGoogle Scholar
  64. 64.
    Kim J, Kaminsky A, Summitt J, Thayer W. New innovations for deep partial-thickness burn treatment with ACell MatriStem matrix. Adv Wound Care (New Rochelle). 2016;5(12):546–52. Scholar
  65. 65.
    Kruper G, VandeGriend Z, Lin H, Zulani G. Salvage of failed local and regional flaps with porcine urinary bladder extracellular matrix aided tissue regeneration. Case Rep Otolaryngol. 2013;2013:917183. Scholar
  66. 66.
    Badylak SF, Freytes DO, Gilbert TW. Extracellular matrix as a biological scaffold material: Structure and function. Acta Biomater. 2009;5(1):1–13. Scholar
  67. 67.
    Sasse K, Brandt J, Lim D, Ackerman E. Accelerated healing of complex open pilonidal wounds using MatriStem extracellular matrix xenograft: nine cases. J Surg Case Rep. 2013;2013(4):rjt025. Scholar
  68. 68.
    Cooley J. Use of porcine urinary bladder matrix in hair restoration surgery applications. Hair Transp Forum Int. 2001;21(3.) Accessed 21 Feb 2017.
  69. 69.
    MatriStem Instructions for Use. Columbia: ACell; 2012.Google Scholar
  70. 70.
    Wang J, Wan R, Mo Y, Li M, Zhang Q, Chien S. Intracellular delivery of ATP enhanced healing process in full-thickness skin wounds in diabetic rabbits. Am J Surg. 2010;199(6):823–32. Scholar
  71. 71.
    Palikaras K, Tavernarakis N. Intracellular assessment of ATP levels in Caenorhabditis elegans. Bio Protoc. 2016;6(23):e22048.  10.21769/BioProtoc.2048.CrossRefPubMedPubMedCentralGoogle Scholar
  72. 72.
    Jurkowitz-Alexander MS, Altschuld RA, Hohl CM, Johnson JD, McDonald JS, Simmons TD, Horrocks LA. Cell swelling, blebbing, and death are dependent on ATP depletion and independent of calcium during chemical hypoxia in a glial cell line. J Neurochem. 2006;59(1):344–52.CrossRefGoogle Scholar
  73. 73.
    Kalogeris T, Baines C, Krenz M, Korthuis R. Ischemia/reperfusion. Compr Physiol. 2017;7(1):113–70. Scholar
  74. 74.
    Cooper GM. The cell: a molecular approach. In: The mechanism of oxidative phosphorylation. 2nd ed. Sunderland, MA: Sinauer Associates; 2000. Scholar
  75. 75.
    Im M, Hoopes J. Energy Metabolism in healing skin wounds. J Surg Res. 1970;10(10):459–64. Scholar
  76. 76.
    Wang W, Yi X, Ren Y, Xie Q. Effects of adenosine triphosphate on proliferation and odontoblastic differentiation of human dental pulp cells. J Endod. 2016;42(10):1483–9. Scholar
  77. 77.
    Kaufmann A, Musset B, Limberg SH, Renigunta V, Sus R, Dalpke AH, Heeg KM, Robaye B, Hanley PJ. Host tissue damage signal ATP promotes non-directional migration and negatively regulates toll-like receptor signaling in human monocytes. J Biol Chem. 2005;280(37):32459–67.CrossRefGoogle Scholar
  78. 78.
    Faas MM, Saez T, de Vos P. Extracellular ATP and adenosine: the Yin and Yang in immune responses? Mol Aspects of Med. 2017;55:9–19. Scholar
  79. 79.
    Corderio J, Jacinto A. The role of transcription-independent damage signals in the initiation of epithelial wound healing. Nat Rev Mol Cell Biol. 2013;14:249–62. Scholar
  80. 80.
    Studzińska B, Seroka A, Lepicka M, Roszek K, Komosynski M. The increase of adenylate kinase activity in the blood can control aggregation of platelets in coronary or peripheral arterial ischemia. Health. 2010;2(3):246–52. Scholar
  81. 81.
    Huang N, Wang D, Heppel L. Extracellular ATP is a mitogen for 3T3, 3T6, and A431 cells and acts synergistically with other growth factors. Proc Natl Acad Sci USA. 1989;86:7904–8.CrossRefGoogle Scholar
  82. 82.
    Hill L, Gavala M, Lenertz L, Bertics P. Extracellular ATP may contribute to tissue repair by rapidly stimulating purinergic receptor X7-dependent vascular endothelial growth factor release from primary human monocytes. J Immunol. 2010;185(5):2038–3034.CrossRefGoogle Scholar
  83. 83.
    Kanneganti TD, Lamkanfi M, Núñez G. Intracellular NOD-like receptors in host defense and disease. Immunity. 2007;27(4):549–59.CrossRefGoogle Scholar
  84. 84.
    Chien S. Intracellular ATP delivery using highly fusogenic liposomes. Methods Mol Biol. 2010;605:377–91. Scholar

Copyright information

© Springer Japan KK, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Energy Delivery SolutionsJeffersonvilleUSA

Personalised recommendations