Methods for the isolation and 3D culture of dermal papilla cells from human hair follicles

Abstract The dermal papilla is a cluster of mesenchymal cells located at the base of the hair follicle which have a number of important roles in the regulation of hair growth. As a consequence, in vitro models of these cells are widely used to study the molecular mechanisms which underlie hair follicle induction, growth and maintenance. While dermal papilla from rodent hair follicles can be digested prior to cell isolation, the unique extracellular matrix composition found in human dermal papilla renders enzymes such as trypsin and collagenase insufficient for digestion of the dermal papilla into a single cell suspension. As such, to grow human dermal papilla cells in vitro, the papilla has to first be isolated via a micro‐dissection approach from the follicle. In this article we describe the micro‐dissection and culture methods, which we use within our laboratory, for the study of human dermal papilla cells.


| INTRODUCTION
Hair follicle morphogenesis is driven by reciprocal epithelialmesenchymal interactions, with the initiating signal for these interactions thought to arise in the mesenchymal dermis. [1] With parallels to development, the hair follicle mesenchyme in adult skin plays an important role regulating hair cycle transitions, specifically the initiation of the hair growth phase known as anagen, and exit from the resting phase telogen. [2] In an anagen hair follicle, the mesenchyme is constituted of two components: the dermal papilla, a flame shaped structure at the base of the follicle, and the dermal sheath, which wraps around the outside of the follicle. While there is movement of cells between the dermal papilla and dermal sheath, [3,4] the dermal papilla is often the more studied component of the mesenchyme. Not only does it have a role in the initiation of anagen, [2] but also its placement subjacent to the hair matrix during anagen means it can signal to these cells directing their differentiation into the different lineages of the follicle. [5,6] In androgenetic alopecia, more commonly known as male pattern baldness, the hair follicle goes through a process known as miniaturisation [7] during which time its hair shaft becomes smaller. [8] This reduction in the size of the hair shaft is coupled with a reduction in the size of the dermal papilla, [9] which is relevant as the volume of the dermal papilla in whisker follicles is believed to be proportional to the volume of the hair shaft. [10] In androgenetic alopecia, follicles can also become "stuck" in telogen and are unable to re-enter anagen.
While it is known that hair follicle stem cells in miniaturising follicles have a reduced ability to convert into stem cell progenitors, [11] it is not known whether this is due to a lack of signalling from the dermal papilla, or an inability of these cells to respond to signals from the dermal papilla. However, given the importance of the dermal papilla in the initiation and maintenance of anagen, a number of research groups have used in vivo, ex vivo and in vitro models of the dermal papilla to elucidate the key signals within it which control hair growth. This includes transcriptome analysis on dermal papilla, and models assessing the role of the dermal papilla in the regulation of the hair cycle, hair development and specification of hair type. [12][13][14][15][16] It has been long reported that intact dermal papillae microdissected from species such as the rat and guinea pig are able to induce This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. new hair and follicle growth in recipient epithelium after transplantation. [17,18] However, it was not until the 1980s that dermal papilla cells were successfully isolated from rat whisker follicles for growth in vitro. [19] This was followed by a demonstration showing these cells from rat could induce new hair growth in recipient epithelium even after they were grown in culture. [20,21] Later, a similar method utilising a micro-dissection approach was used to isolate and culture human dermal papilla cells. [22] However, while fully intact human papilla can induce new hair growth, [23] cells cultured from these dermal papillae are notably dissimilar to their rodent counterparts and quickly lose their inductive capacity with culture. [24] In their natural environment in vivo, the dermal papilla is found at the base of the hair follicle containing cells surrounded by a proteoglycan-rich extracellular matrix. [25] As mentioned above, human dermal papilla cells lose their ability to induce new hair growth in culture. We previously demonstrated that growth of human dermal papilla cells in hanging drops can promote formation of dermal spheroids, which are morphologically akin to intact papilla. [26] This forced aggregation enables a partial restoration of their inductive memory, and human dermal papilla cells grown in 3D spheroids can induce new hair growth. [24,27] Nowadays, enzymatic digestion followed by fluorescenceactivated cell sorting (FACS) is frequently used for isolation of murine pelage papilla, [28] which are too small to be micro-dissected.
However, the unique extracellular matrix composition of human dermal papillae [25,29] renders them indigestible to a single cell suspension with commonly used enzymes such as trypsin and collagenase. Digestion of the human hair follicle with collagenase will digest everything except the dermal papilla, which remains intact, with only a minor disruption to its extracellular matrix. [30] As such, a variety of micro-dissection approaches and explant culture methods are still used to isolate dermal papilla from human scalp follicles. [31,32] In this article, we provide a detailed methodology on the inversion microdissection technique which we use within our laboratory to isolate dermal papilla cells from human scalp follicles in anagen, as well as some tips on how to avoid common pitfalls. In addition to this, we also detail a method for dermal papilla 3D spheroid formation.
Previously, low binding plates or plates with synthetic membranes have been used to generate dermal papilla spheroids; [27,33] however, here we describe a hanging drop methodology which we use within our laboratory. [26] 2 | TECHNIQUES

| Isolation of human dermal papillae
To obtain human tissues, appropriate ethical approvals should be in place. We use skin samples from patients who have given their informed consent using ICREC-approved consent forms. Tissue is stored under Human Tissue Authority licence 12275 of the Imperial College Human Tissue Bank. Skin biopsies are often full thickness and therefore have an epidermis, dermis and dermal white adipose tissue, with the base of the hair follicles located in the adipose tissue. If the biopsy is from the scalp, then approximately 90% of these follicles will be in anagen. [34] The dermal papilla can be identified using a stereomicroscope at a high magnification (Leica M80 on a TL 5000 ergo base). As the dermal papilla is engulfed within the hair matrix, we use an inversion technique for isolation. This dermal papilla cell isolation method can be divided into the following three parts, which are also shown in a Video S1.
Following this, transfer the skin biopsy to a new petri dish containing DMEM supplemented with 1% ABAM ( Figure 1A).
B. While the tissue is washing, prepare plates for use later in the protocol. Using a sterile Pasteur pipette, put eight small drops, and one larger drop of DMEM supplemented with 1% ABAM onto the inverted lid of a petri dish ( Figure 1B). Each small drop will be used to hold a single end bulb for inversion in Part 2. Cover these drops by placing the base of the petri dish inside the lid.
C. If required, section the tissue into manageable pieces using a sterile scalpel. However, be careful not to cut any hair follicles in half.
Using sterile Noyles spring scissors and Dumont forceps, trim away any adherent adipose or connective tissue surrounding the lower section of the follicle to expose the end bulb located at the base of the follicle. The forceps can be used to gently pull up connective tissue and adipose tissue into a peak, away from each follicle, while the scissors can be laid parallel to each follicle to clean efficiently. Tip 1: Cleaning may not always be required if the biopsy contains exposed follicles, as shown in the Video S1.
D. Using sterile Dumont straight tip forceps, secure the hair follicle in place so the dermal papilla and hair matrix are visible. Using a pair of scissors, transect the follicle through the matrix just above the papilla to isolate the end bulb ( Figure 1C). Carefully transfer the end bulb on the end of the scissors to a clean petri dish containing drops of DMEM/1%ABAM that was prepared in Part 1B ( Figure 1B).
Drops of DMEM are placed on the lid of the petri dish rather than the base to ensure the optimal angle of inversion, as described in Part 2. After cutting the number of end bulbs required, proceed to the inversion step. Note 1: To establish a culture in a 35-mm dish, we usually use eight end bulbs distributed in eight drops of DMEM.
The larger remaining drop of DMEM is used to collect inverted end bulbs together before isolation of papillae.

| Dermal papilla spheroid culture
One limitation with culturing human dermal papilla cells to study hair inductivity is that they lose their inductivity quickly in culture. [24] However, when dermal papilla cells are grown in 3D dermal spheroids structures, they partially regain their intact transcriptional signature and associated inductivity. [24] Below, we describe how to prepare 3D spheroids using a hanging drop methodology.
For this, we used dermal papilla cells at passage 3-5, isolated as in the method above. We have divided the procedure into the following two parts: B. Dermal papilla cells grown in antibiotics will not form spheres using the hanging drop method. When cells are being passaged as in Part 1A, start using antibiotic-free medium. If cells are not to be passaged, still change the medium to antibiotic-free medium at least 48 hours before spheroid culture.

| FUTURE PERSPECTIVES
While the skin and hair follicle are perhaps the most accessible tissues in the human body, certainly as compared to other internal tissues and organs, the location of the dermal papilla within the follicle can make isolation by micro-dissection difficult and time-consuming. Subsequently, current practice for the isolation of human dermal papilla is commonly reliant upon a skillset in micro-dissection. New methods of human dermal papilla cell isolation, capable of reducing complexity and time, or increasing cell yield, would be highly advantageous to the skin and hair follicle biology field. The identification of dermal papilla cell-specific surface markers through the analysis of transcriptome data [16,24,35] could potentially allow for the use of high-throughput cell sorting systems, such as FACS or magnetic-activated cell sorting (MACS). In other reports, dermal fibroblasts from the reticular dermis were specifically sorted using the MACS technique and used for bone regenerative applications. [36,37] Despite our increased knowledge of papilla-specific markers, the main limitation with these approaches is that human dermal papilla cannot be In terms of cell division, proliferation ceases when papilla cells are grown in spheroids. [26] We believe this makes spheroid culture a superior model compared to cells in 2D culture, if the questions being asked relate to intact papilla behaviour. This observation, that 3D cell models are superior to 2D cultures, was suggested many years ago in the cancer research field, [38] and subsequent to this, the use of 3D tumor models has accelerated biomarker and drug discovery. [39] That being said, the hanging drop method for cell culture can be arduous and time-consuming. Drug screens require large number of cells to complete, which is not always possible to achieve using primary dermal papilla cells.
To summarise, here we have detailed methods on human dermal papilla isolation, and 3D spheroid culture. The dermal papilla is an in-