Las in vitro


Polona Tratnik

Hair in vitro (2010) is a part of the four-years lasting transdisciplinary research project, within which a team of collaborators cultivate hair in vitro. After getting a human tissue sample with hair from a donor by a plastic surgery, they execute series of experiments in reaching a maximally long lasting life period of the artificially cultivated biomaterial, separated from the human body in the highly controlled laboratory. The liveness is being proved with visualization in time. For this purpose they perform monitoring with sequence photographing of the sample that shows how the observed cells divide and form the hair form. Experiments include elimination of the stem cells from the samples and testing the influence of stem cells on the culture medium with hair and the belonging skin cells.

The project is rhizomatically structured at several levels and connects technoscience with heterogeneous artistic strategies and with humanistic research in tissue engineering and immunology as socially especially actual fields of biotechnology that promise revolutionary consequences, especially in medicine and aesthetics surgery. The project as well reflects the hybridization of art, humanities and technoscience, which is today at slope. The team of authors-executors is focused on research process and on consistent connecting fields, harmonizing the heterogeneous interests. The work is not oriented to producing finished products, artifacts for observer's contemplation, but to opening of the research process and the whole discourse to the public at diverse occasions. The aims of the project are communication of biotechnological potentials with wider public, realization of specific goals, which are interesting from the biotechnological, artistic and other aspects, and discussing the related issues, which are extremely important for contemporary individual and society.

»The medium used in a situation where manipulation of living material in vitro takes place in the real time, is not a medium that would assure an image anymore, and it is even not a medium in which life would be embodied, but is a medium as a replacement of bodily environment with its entire wetness, temperature and other fragility, which conditionally, if an experiment is successful, enables the survival of a cell for a certain period. Life being constantly endangered by death, is here present in an extreme sensitivity and can stop in any moment; however its ending will externally not be present as a boundary, line, break, but has been constantly present as suspended for an indefinite period in the future and in the past as well; likewise in the ending life is present as a possibility, with which one can manipulate with a help of biotechnology.«

»Artistic situations that repeat the gesture of mastery and control of life however do not repeat the social tendency to systematic attainment of absoluteness but on the contrary: they open the social body in its core, with establishing the rhizomatic body they immersively (here and now, inside and outside) position the subject into a nonorientational environment-body, in which the subject becomes a schizophrenic cell that becomes aware of the schizophrenic self-society in an affective confrontation.«

»In the planomenons, planes of consistencies or compositions of such a transdisciplinary hybrid, which are not hierarchically organized any more, once predominantly biotechnological, secondly artistic, thirdly humanistic, fourthly some other colonies condense, however none of them is ever purified of the others, elements of one have become the satellites of the other. It has become impossible to distinguish such colonies since they intertwine and function simultaneously in mutual reconciliation.«

doc. dr. Polona Tratnik, In vitro.
Živo onstran telesa in umetnosti,
Koper: Univerza na Primorskem, 2010

»To grow hair in vitro and then actually use it in aesthetic surgery I find almost hard to believe. But hair in cultures will help us to learn more about the whole physiology of hair, its way of reproduction and how certain substances affect hair. This means that hair is a model for discovering and testing new medicines. This is also the goal of our research project which is supported by Slovenian Research Agency. The goal that we want to achieve is to determine how can cells from hair follicle contribute to the whole group of useful stem cells. This means a new source of cells that can then be used in other parts of regenerative medicine.«

»I decided to cooperate mostly because of the observation that there is a big void between contemporary science and everyday life. I see that some people expect too much from science or they have a distorted picture, while scientists often believe that they have to function in a closed environment where the communication is confined. When information leave the closed environment it is with excessive enthusiasm or more oftenwith an excessive affair. When the latter happens the damage is hard to repair. With art the science can communicate with people and get their response in a different way. That is on emotional level, on level of impressions, reactions which an artist can easier and more sensitive detect then a scientist with his experimental methods.«

doc. dr. Miomir Knežević, univ. dipl. biol.,
Hair In Vitro, 15th June 2010. © Horizonti.

»Our story of transplanting and growing hair may not be directly applicable but it is very instructive. Every such project must unite the whole chain of experts and much diversified knowledge is needed. With growing some preparation the surgical part is smaller, is in withdrawal and in some basic understanding of tissue. Afterwards comes the growing of tissue, certain preparations etc. These are the whole chains of people and their knowledge. The thing that is interesting is cooperation and multidisciplinarity, is to walk over your own limited condition, into which you can quickly fall and that you create something new and good.«

»Art probably meets with serious science in that, that they both originate from the same roots. Both arise from some deep human search. Not only art is beautiful, the real science can also be. Mathematics is beautiful. It has some transcendence. Such work, playing, researching as we find in science, we probably also find in art. In art are also people which sacrifice their life, that are willing to give up something in order to achieve something, to dive somewhere. We just call something science, something art. Now that there are so many possibilities things start to intertwine. Soul mates find each other and something is born.«

Aleš Leskovšek, Dr. Med.,
Spec. Plast., Rec., Aesthet. Surgery,
Hair In Vitro, 15th June 2010. © Horizonti.

 

Quotations

THE BIOLOGY OF HAIR FOLLICLES

»Hair has many useful biologic functions, including protection from the elements and dispersion of sweat-gland products (e.g., pheromones). It also has psychosocial importance in our society, and patients with hair loss (alopecia) or excessive hair growth often suffer tremendously.« p491

HAIR FOLLICULAR CELL/ORGAN CULTURE IN TISSUE ENGINEERING AND REGENERATIVE MEDICINE

»Hair follicles are complex organs composed of the dermal papilla (DP), dermal sheath (DS), outer root sheath (ORS), inner root sheath (IRS) and hair shaft. Development of hair follicles begins towards the end of the first trimester of pregnancy and is controlled by epidermal–mesenchymal interaction (EMI), which is a signaling cascade between epidermal and mesenchymal cell populations. Hair grows in cycles of various phases.« p323

»Conversely, the transplantation of human hair involves taking plugs of natural hair from areas in which occipital hair is growing and transplanting them to bald areas. However, the number of hairs that can be transplanted is limited in that only three such operations can generally be performed. To overcome such problems, many researchers have attempted to revive hair follicles by culturing hair follicle cells or mesenchymal cells in vitro and then implanting them in the treatment area.« p323

»A hair follicle is part of the skin that grows hair by packing old cells together. Sebaceous glands, which are tiny sebum-producing glands found everywhere except on the palms, lips and soles of the feet, are attached to each hair follicle. The thicker the density of hair, the more sebaceous glands are found. Stem cells are located at the junction of the sebaceous gland and the follicle and are primarily responsible for ongoing hair production via a process known as the anagen stage.« p324

»Hair follicles are divided into two major classes that occur in epidermal tissues and dermal tissues.« p324

»Cyclosporin A (CsA) has been used as a potent immunosuppressive agent for inhibition of graft rejection following organ transplantation.« p327

»Specifically, when hair follicles were cultured without insulin, they induced rapid transformation from anagen to catagen.« p327

»Substrates such as nylon mesh seeded with skin fibroblasts and full thickness skin were also found to support the growth of hair.« p327

»The evolution of hair follicle engineering began with the recognition in the early 1960s that hair follicles could be transplanted clinically into a foreign site and still grow a shaft typical of the donor site.« p329

SIMPLE AND RAPID METHOD TO ISOLATE AND CULTURE FOLLICULAR PAPILLAE FROM HUMAN SCALP HAIR FOLLICLES

»Published methodologies of HFP isolation rely on microdissecting manipulation for their successful isolation.« p381

»Moreover, imprecise manipulations at the level of the lower connective tissue sheath may increase the probability of dislodging and subsequently aspirating cells from the lower connective tissue sheath and/or epithelium. These cells may go on to contaminate the HFP culture.« p382

»Hair follicle papillae were individually isolated from intact isolated HF under bright-field stereoscopic illumination.« p382

»The quality of the HFP isolated using this method was assessed by high-resolution light microscopy (HRLM) and transmission electron microscopy.« p384

»Cultures of HFP are primarily obtained from terminal HFs.« p384

HUMAN HAIR GROWTH IN VITRO

»Human anagen hair follicles were isolated by microdissection from human scalp skin. Isolation of the hair follicles was achieved by cutting the follicle at the dermo-subcutaneous fat interface using a scalpel blade. Intact hair follicles were then removed from the fat using watchmakers' forceps. Isolated hair follicles maintained free-floating in supplemented Williams E medium in individual wells of 24-well multiwell plates showed a significant increase in length over 4 days.« p463

»Hair growth, which is effected by the division of the hair follicle matrix cells under control of the dermal papilla, is cyclical in the mammal.« p463

»These data show that in vitro hair follicles are able to maintain the in vivo pattern of DNA synthesis, and so it is reasonable to suppose that the production of a keratinised hair shaft in the maintained hair follicle occurs as a result of matrix cell division in the hair follicle bulb.« p468

HUMAN HAIR CYCLE

»The length of hair cycles in man varies in the different regions of the body.« p65

»Methods of measuring human hair cycles: calculation of the length of the cycle by measuring the final length of hair; marking the tips of individual hairs with ink dye; plucking hairs and observing their roots to determine the cycle periods.« p65

HAIR IN FORENSIC MEDICINE

»Possible applications of hair analysis include diagnosis of drug abuse, poisoning and doping, evaluation of occupational exposure to toxics, evaluation of prenatal exposure to drugs, and monitoring of a patient's compliance with drug prescription.« p539

»Drugs and toxics are incorporated into hair through three different modalities:
Passive diffusion to the hair matrix from the blood and successive incorporation in the hair shaft during keratinization. Transfer to the formed hair shaft from sebum and sweat. Transfer to the formed shaft from the environment.« p540

»Hair sampling is usually made from the vertex or from the pubis where hair is less contaminated by environmental and cosmetic factors.« p541

HAIR GROWTH ASSESSMENT TECHNIQUES

»Qualitative and quantitative methods are essential for objectively evaluating hair growth activity.« p125

CYCLOSPORIN A-INDUCED HAIR GROWTH IN MICE IS ASSOCIATED WITH INHIBITION OF CALCINEURIN-DEPENDENT ACTIVATION OF NFAT IN FOLLICULAR KERATINOCYTES

»Immunosuppressive immunophilin ligands such as cyclosporin A (CsA) and FK506 are known as potent hair growth modulatory agents in rodents and humans that induce active hair growth (anagen) and inhibit hair follicle regression (catagen).« p1593

»The immunosuppressive effectiveness of these drugs has been generally attributed to inhibition of T cell activation through well-characterized pathways.« p1593

»Growth and development of hair follicles are influenced by a variety of growth factors and cytokines, the most widely described being keratinocyte growth factor (KGF), interleukin-1 (IL-1), and transforming growth factor _ (TGF_).« p1594

AT THE ROOTS OF A NEVER-ENDING CYCLE

»Wnt pathways in the development of many different organs in the body, including limbs, cartilage, kidney, and brain (reviewed by Cadigan and Nusse, 1997).« p18

»While normal dermal papilla cells rapidly lose their inductive powers in culture (Jahoda et al., 1984), the Wnt-bathed dermal papilla cells maintained inductive capacity to generate hair follicles when trans- are planted back into mice.« p19

»A discussion of the biochemical events involved in Wnt signaling and hair follicle formation would not be complete without further discussion of the Shh signal transduction pathway.« p22

ANIMAL MODELS AND CULTURE METHODS IN THE STUDY OF HAIR GROWTH

»Further, if each follicle contains the clock signal, then a growth wave must arise from coordination of individual follicles by an overriding signal, presumably of endocrine origin.« p105

»In a recent discussion of hair cycling,2 an inhibitor theory was proposed in which the removal or release of an inhibitory molecule in the epithelium stimulates anagen and growth; however, this leads to the question as to what is the nature of the event that removes the inhibitor.« p105

»Culture systems should become increasingly important in the dissection of follicle cell function in hair diseases.« p110

»The important interactions known to occur between the epithelial and mesenchymal components of skin can best be studied using in vitro models for all of the above reasons; however, there are also some potential disadvantages of in vitro models that should not be ignored.« p110

CHARACTERIZATION OF A NEW TISSUE-ENGINEERED HUMAN SKIN EQUIVALENT WITH HAIR

»This model was produced exclusively from human fibroblasts and keratinocytes and did not contain any synthetic material.« p318

»Therefore, this new hairy, human skin equivalent mode/allowed an experimental design in which the only variable was the presence of pilosebaceous units and provided new data confirming the importance of hair follicles in pereutaneous absorption.« p318

»Hair follicles have been previously cultured in vitro, in an organ culture system, or in sponge-supported histoculture (Philpott et al., 1990; Li et al. 1992).« p318

»Hair follicles have also been used as a source of keratinoeytes in a skin equivalent model (Lenoir et al., 1988; Lenoir-Viale et al., 1993).« p318

»Hairless animals are not reliable models because they still have hair follicles.« p319

»Human keratinocytes and dermal fibroblasts were obtained from normal adult skin specimens removed during reductive breast surgery.« p319

»Human fibroblasts were obtained from the dermal portion of skin biopsies.« p319

»Control samples (skin equivalents with sham hair insertion) were obtained by laying down an explant of interfollicular epidermis (i.e., without hair follicle) on top of the holes previously made in the dermal equivalent. An additional fibroblast sheet was also added under the dermal equivalent.« p319

»For histological analysis, skin equivalents were fixed with Bouin's solution and paraffin embedded. Six-lam-thick sections were stained with hematoxylin, phloxine, and saffron.« p319

THE BIOLOGY OF HAIR GROWTH

»Hair follicles go through well established repeated cycles of development and growth (anagen), regression (catagen), and rest (telogen) to enable the replacement of hairs, often by another of differing colour or size. An additional phase, exogen, has been reported where the resting club hair is released.« p10

1. Green M.R., Clay C.S., Gibson W.T., Hughes T.C., Smith C.G., Westgate G.E., White M. in Kealey T. 1986. Rapid Isolation in Large Numbers of Intact, Viable, Individual Hair Follicles From Skin: Biochemical and Ultrastructural Characterization. J Invest Dermatol, 87:768-770

2. The strip of skin was repetedly cut with a loose-fitting pair pf scissors for 1-3 m and the supernatant liquid removed and examined under Wild M8 binocular dissecting microscope. Str.768

3. Keug P. J., Hoon L.D., Hoon Y.H., Ho S.Y., Yong S.K. 2006. Method for the preparation of a dermal papilla tissue having hair follicle inductive potency. KR20040098680 20041129 – PATENT Živa

4. Philpott M.P., Green M.R. in Kealey T. 1989. Studies on the biochemistry and morphology of freshly isolated and maintained rat hair follicles. Journal of Cell Scienc 93, 409-418
We now show that these follicles can be viably maintained on permeable supports for 7 days in vitro as determined by their adenine nucleotide contents, rates of [mefliy/-3H]thymidine and [U-14C]leucine uptake, [methyl- H]thymidine autoradiography, patterns of keratin synthesis and light and electron microscopy. Str. 409

5. Philpott M.P., Green M.R. in Kealey T. 1990. Human hair growth in vitro. Journal of Cell Scienc 97, 463-471
We report for the first time the successful maintenance and growth of human hair follicles in vitro.
Isolated hair follicles maintained free-floating in supplemented Williams E medium in individual wells of 24-well multiwell plates showed a significant increase in length over 4 days. Str. 463

Richardson G.D., Arnott E. C., Whitehouse C. J., Lawrence C. M., Hole N., in Jahoda C.A.B. 2005. Cultured Cells from the Adult Human Hair Follicle Dermis can be Directed Toward Adipogenic and Osteogenic Differentiation. J Invest Dermatol, 124:1090-1091
Here, we report adipogenic and osteogenic differentiation in human hair follicle-derived DP and DS cultures. Str 1090

Kim1 J.Y.,, Tavaré S. in Shibata D. 2006. Human hair genealogies and stem cell latency. BMC Biology 2006, 4:2
Average hair errors increased until two years of age, and then were constant despite decades of replacement, consistent with new hairs arising from infrequently dividing bulge stem cells. Errors were significantly more frequent in longer hairs, consistent with long-lived but eventually shed mitotic follicle cells. Str. 1

Lyle S., Christofidou-Solomidou M., Liu Y., Elder1 D.E., Albelda S. in Cotsarelis G. 1998. The C8/144B monoclonal antibody recognizes cytokeratin 15 and defines the location of human hair follicle stem cells. Journal of Cell Science 111, 3179-3188
The homeostasis of all self-renewing tissues, including the epidermis and hair follicle, is thought to be dependent on stem cells As undifferentiated stem cells divide, they generate daughter cells that retain the stem cell phenotype, and daughter cells (called transit-amplifying (TA) cells) that undergo rapid proliferation and terminal differentiation to repopulate the tissue str. 3179

Porter R.M. 2003. REVIEW: Mouse models for human hair loss disorders. J. Anat. 202, 125–131
Changes in hair growth str. 127

Spradling A., Drummond-Barbosa D. in Toshie Kai. 2001. insight review articles: Stem cells find their niche. Nature vol. 414., 98-104
Hair follicles contain two zones of stem-like cells. The hair shaft and its surrounding sheaths are produced by matrix cells located on a basement membrane overlying the protruding piece of dermis know as the dermal papilla. Str. 101

Jones C.J. in Kealey T. 1987. Electrophysiological and dye-coupling studies on secretory, myoepithelial and duct cells in human eccrine sweat glands. J.Physiol. 389, 461-481

LEE C.M., JONES C.J. in Kealey T. 1984. Biochemical and ultrastructural studies of human eccrine sweat glands isolated by shearing and maintained for seven days. J.Cell Sci. 72, 259-274
Sweat glands were picked out using two pairs of stainless steel Dumont no. 5 microforceps. Str. 206

 

Articles

Alonso L., Fuchs E. 2006. The hair cycle. Journal of Cell Science, 119, 391–393.

Blume-Peytavi U., Hillmann K., Guarrera M. 2008. Hair growth assessment techniques. Hair growth and disorders. Whiting D. A., Trϋeb R. M. (eds.). Leipzig, Springer, 125–157.

Fuchs E., Merrill B.J., Jamora C., DasGupta R. 2001. At the roots of a never-ending cycle. Developmental cell, 1, 13–25.

Gafter-Gvili A., Sredni B., Gal R., Gafter U., Kalechman Y. 2003. Cyclosporin A-induced hair growth in mice is associated with inhibition of calcineurin-dependent activation of NFAT in follicular keratinocytes. AJC Cell Physiology, 284, 1593–1603.

Green M.R., Clay C. S., Gibson W. T., Hughes T. C., Smith C. G., Westgate G. E., White M., Kealey T. 1986. Rapid Isolation in Large Numbers of Intact, Viable, Individual Hair Follicles From Skin: Biochemical and Ultrastructural Characterization. J Invest Dermatol, 87, 768–770.

Jones C. J. in Kealey T. 1987. Electrophysiological and dye-coupling studies on secretory, myoepithelial and duct cells in human eccrine sweat glands. J. Physiol. 389, 461–481.

Keug P. J., Hoon L. D., Hoon Y. H., Ho S. Y., Yong S. K. 2006. Method for the preparation of a dermal papilla tissue having hair follicle inductive potency. KR20040098680 20041129.

Kim1 J. Y.,, Tavaré S. in Shibata D. 2006. Human hair genealogies and stem cell latency. BMC Biology 2006, 4, 2.

Lee C. M., Jones C. J., Kealey T. 1984. Biochemical and ultrastructural studies of human eccrine sweat glands isolated by shearing and maintained for seven days. J.Cell Sci. 72, 259–274.

Lyle S., Christofidou-Solomidou M., Liu Y., Elder1 D.E., Albelda S. in Cotsarelis G. 1998. The C8/144B monoclonal antibody recognizes cytokeratin 15 and defines the location of human hair follicle stem cells. Journal of Cell Science 111, 3179–3188.

Magerls M., Kauser S., Tobin P. R. 2002. Simple and rapid method to isolate and culture follicular papillae from human scalp hair follicles. Experimental Dermatology, 11, 381–385.

Michel M., L'heureux N., Pouliot R., Xu W., Auger F. A., Germain L. 1999. Characterization of a new tissue-engineered human skin equivalent with skin. In Vitro Cellular & Developmental Biology-Animal, 35, 6, 318-326

Millar S. E., Willert K., Salinas P. C., Roelink H., Nusse R., Sussman D. J., Barsh G. S. 1999. WNT signaling in the control of hair growth and structure. Developmental Biology, 207, 133–149.

Morioka K. 2005. Hair Follicle: Differentiation under the electron microscope. Tokio, Springer, 150.

Paus F., Cotsarelis G. 1999. Mechanisms of disease: The biology of hair follicles. The New England Journal of Medicine, 341, 7, 491–497.

Peus D., Pittelkow M. 1996. Growth factors in hair organ development and the hair growth cycle. Dermatologic Clinics, 14, 4, 559–572.

Philpott M., Green M. R., Kealey T. 1990. Human hair growth in vitro. Journal of Cell Science, 97, 463–471.

Philpott M. P., Green M. R. in Kealey T. 1989. Studies on the biochemistry and morphology of freshly isolated and maintained rat hair follicles. Journal of Cell Science 93, 409–418.

Porter R. M. 2003. REVIEW: Mouse models for human hair loss disorders. J. Anat. 202, 125–131.

Piraccini B. M., Pazzaglia M., Tosti A. 2008. Hair in forensic medicine. Hair growth and disorders. Whiting D.A., Trϋeb R.M. (eds.). Leipzig, Springer, 539–542.

Randall V. A., Botchkareva N. V. 2009. The biology of hair growth. Cosmetic applications of laser and light-based systems. UK, William Andrew Inc., 3–35.

Rogers G., Hynd P. 2001. Animal models and culture methods in the study of hair growth. Clinics in Dermatology, 19, 105–119.

Richardson G. D., Arnott E. C., Whitehouse C. J., Lawrence C. M., Hole N., in Jahoda C. A. B. 2005. Cultured Cells from the Adult Human Hair Follicle Dermis can be Directed Toward Adipogenic and Osteogenic Differentiation. J Invest Dermatol, 124, 1090–1091.

Shimomura Y., Zlotogorski A., Christiano A.M. 2008. Molekular genetics of human hair diseases. Hair growth and disorders. Whiting D.A, Trϋeb R.M. (eds.). Leipzig, Springer, 85–100.

Spradling A., Drummond-Barbosa D. in Toshie Kai. 2001. insight review articles: Stem cells find their niche. Nature vol. 414., 98–104.

Yoo B., Shin Y., Yoon H., Seo Y., Park J. 2009. Hair follicular cell/organ culture in tissue engineering and regenerative medicine. Biochemical Engineering Journal, 48, 323–331.

horizonti.net

Team: doc. dr. Polona Tratnik (project leader, art and humanities), doc. dr. Miomir Knežević (biotechnology leader), prof. dr. Primož Rožman (biotechnology), Ajda Marič (biotechnology), Živa Marinko (biotechnology), Aleš Leskovšek, dr. med., spec. plast., rec., aesthet. surgery (plastic surgery), Miha Turšič (visual communication), Damjan Švarc (photography), mag. Robi Černelč (film director), Jože Baša (video production), prof. dr. Miško Šuvaković (art theory and philosophy), assist. Maja Murnik (phenomenology), doc. dr. Katja Kolšek (biopolitics), María Antonia González Valerio (philosophy), Jens Hauser (curator), Jurij Krpan (curator).

Producer: Horizonti – Institute for Art, Culture, Science and Education. Co-Producers: Blood Transfusion Centre of Slovenia ~ University of Primorska, Science and Research Centre of Koper.

Project support: Ministry of Culture of Slovenia ~ Slovenian Research Agency ~ Educell d. o. o. ~ Tehnooptika Smolnikar d. o. o. ~ Kapelica Gallery ~ Museum of Modern Art Ljubljana ~ University of Ljubljana, Medical Faculty ~ SIMED Zdravstvo d. o. o. ~ INCUBATOR: Hybrid Laboratory at the Intersection of Art, Science and Ecology, School of Visual Art, University of Windsor ~ Universidad Nacional Autónoma de México, Facultad de Filosofía y Letras ~ Center of Digital Arts, School of Social Sciences and Humanities, University of Science and Technology of China ~ Artist's Association of Tampere ~ ARTE Television ~ EuroNews.