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Volume 15, Issue 3-4

Issues

Laser-based 3D cell printing for tissue engineering

Lothar Koch / Andrea Deiwick / Boris Chichkov
Published Online: 2014-11-12 | DOI: https://doi.org/10.1515/bnm-2014-0005

Abstract

Currently, different 3D printing techniques are investigated for printing biomaterials and living cells. An ambitious aim is the printing of fully functional tissue or organs. Furthermore, for manifold applications in biomedical research and in testing of pharmaceuticals or cosmetics, printed tissue could be a new method, partly substituting test animals. Here we describe a laser-based printing technique applied for the arrangement of vital cells in two and three-dimensional patterns and for tissue engineering. First printed tissue, tested in vitro and in vivo, and printing of cell patterns for investigating cell-cell interactions are presented.

Keywords: bioprinting; cell array; cell printing; laser induced forward transfer; tissue printing

Introduction

For engineering complex tissue as well as for (stem-)cell-based therapies, a fundamental understanding of interactions between different cells and their environment is essential. Common ex vivo cell studies in two-dimensional cell cultures are not appropriate to simulate the complex interactions in 3D tissue and cell-microenvironments in vivo. Cell behavior differs dramatically in 3D.

Thus, printed 3D cell patterns could bridge the gap between common cell culture conditions in vitro and animal models. Innovative 3D cell models could provide new insights in cell behavior, tissue functions and regeneration, and the effect of agents like pharmaceuticals or cosmetics ex vivo.

Different techniques are developed for generating specific cell patterns, commonly called “bioprinting” or “cell printing” techniques. The most prevalent cell printing techniques are ink-jet printing technology, extrusion technology, and laser-assisted bioprinting (LaBP) based on the so-called laser-induced forward transfer. They have different advantages and disadvantages and may be preferred for specific applications.

For printing cells with these techniques, the cells are embedded in hydrogel precursors, so-called sols, or hydrogels and deposited (printed) in a droplet or strand. For printing 3D cell patterns or tissue, the hydrogel needs to fulfil several requirements. First, it has to be suitable for the printing process. Thereby, a shear thinning nature and an adjustable viscosity is advantageous. Second, it has to support cell survival. Third, it needs to be adequate as the extra-cellular matrix of the projected cell pattern or tissue. Fourth, it should not affect cell behavior in an unintended way. Therefore, often a sol is printed with the embedded cells and then gelled with a cross-linker to yield appropriate stiffness for a 3D structure.

Examples are fibrinogen mixed with hyaluronic acid and cross-linked with thrombin or blood plasma mixed with alginate and cross-linked with calcium chloride. Cross-linkers were printed as a further layer or sprayed onto the printed sol. Alternatively, the sol was printed into a cross-linker reservoir [1].

However, some gels like collagen, the most abundant protein in mammals, do not allow a post-print gelling, if cells are embedded. The gelling of collagen occurs by a change of the pH value. Since cells do not survive in acidic or basic sols, a complete mixture of the gel before printing is required. Thus, the ability of printing high viscosity gels is needed.

LaBP is capable of high resolution printing of cells, embedded in hydrogels, with high cell densities and a wide range of rheological properties. The volume of the printed droplets can be varied easily in a wide range from some picoliters to nanoliters and the amount of cells per droplet can be varied from single cells to tens of cells [2, 3].

A schematic sketch of the printing setup is shown in Figure 1A. In principle, a glass slide or laser-transparent ribbon is coated with a thin layer of a laser absorbing material, e.g., gold, titanium, polymers like triazene [4], or gelatin [5]; also assemblies with two layers [6, 7] or without absorbing layer ([8]; here, the cell-containing sol acts as the laser absorbing material) have been tested.

(A) Schematic sketch of the laser-assisted bioprinting between two coplanar glass slides: The upper slide is coated underneath with a laser absorbing layer and a layer of biomaterial to be transferred. Laser pulses are focused through the upper glass slide into the absorbing layer. By evaporating this layer a high gas pressure is generated, that propels the biomaterial towards the lower glass slide. (B) Stroboscopic images of the printing process between two glass slides taken with different time delays with respect to the laser pulse. The distance between the glass slides is ∼500 μm. The dark horizontal lines appear due to interference at the two glass slides. Shortly after the laser pulse impact, a growing bubble develops, driven by the expanding vaporized absorption layer. This bubble collapses without reaching the lower glass slide after a few microseconds, forming a liquid jet. This jet lasts for some hundred microseconds, reaches the lower glass slide and remains there as a droplet. (C) Droplets of hyaluronic acid and fibrinogen sol containing NIH3T3 fibroblasts, printed on a fibrin layer. The distance between two neighboring droplets is 600 μm.
Figure 1

(A) Schematic sketch of the laser-assisted bioprinting between two coplanar glass slides: The upper slide is coated underneath with a laser absorbing layer and a layer of biomaterial to be transferred. Laser pulses are focused through the upper glass slide into the absorbing layer. By evaporating this layer a high gas pressure is generated, that propels the biomaterial towards the lower glass slide. (B) Stroboscopic images of the printing process between two glass slides taken with different time delays with respect to the laser pulse. The distance between the glass slides is ∼500 μm. The dark horizontal lines appear due to interference at the two glass slides. Shortly after the laser pulse impact, a growing bubble develops, driven by the expanding vaporized absorption layer. This bubble collapses without reaching the lower glass slide after a few microseconds, forming a liquid jet. This jet lasts for some hundred microseconds, reaches the lower glass slide and remains there as a droplet. (C) Droplets of hyaluronic acid and fibrinogen sol containing NIH3T3 fibroblasts, printed on a fibrin layer. The distance between two neighboring droplets is 600 μm.

A layer of the cell-containing sol is blade coated on top of the absorbing layer. Then, the glass slide is mounted upside-down above the item onto which the cells shall be printed. This may be a second glass slide (probably coated with a hydrogel layer), a scaffold [9], or a valvular leaflet [10].

Through the glass slide, the laser pulses are focused into the absorption layer, which is rapidly evaporated in the focus. A vapor bubble expands and propels the subjacent sol downwards. As depicted in Figure 1B, the vapor bubble reaches its maximum size within a few microseconds and re-collapses, when the inner pressure decreases below the outer pressure. Nevertheless, the accelerated sol moves on downwards by inertia, forming a thin jet that lasts for some hundred microseconds. As a result, a volume of some picoliters up to several nanoliters remains as a droplet [11]. The printed droplets’ volume depends on laser pulse energy, the thickness of the absorption layer as well as the thickness and viscosity of the initial sol layer [2].

The quantity of cells per droplet is usually depending on the cell density in the initial sol layer and the droplet’s volume and is subject to statistical variations. Alternatively, droplets containing single cells might be printed, but this requires a low cell density and is time consuming, since each cell needs to be targeted separately. By moving the laser beam and the glass slide, droplets are deposited in two-dimensional patterns (Figure 1C); three-dimensional patterns can be generated layer-by-layer.

There are some differences between the realizations of LaBP with respect to the material, in which the laser radiation is absorbed, and the applied laser system. A detailed description of these different realizations and denominations are given by Schiele et al. [4].

Successful cell printing was reported by several independent groups with various kinds of cells. For applying bioprinting techniques, it is crucial that cells are not affected by the printing process. This implies that they keep their vitality and behavior, their phenotype, and genotype, and that stem cells keep their differentiation potential. Hence, the impact of the printing process on cells and especially stem cells was extensively investigated. Here, this is only summarized briefly: Several groups [12–14] reported near 100% post printing cell viability with several types of absorption layers and cell types. It was demonstrated [14, 15] that DNA strand breaks are not increased, and thus, the genotypes of the cells remain unaffected by printing. Koch et al. [14] reported in addition no increase in apoptosis of cell lines and human mesenchymal stem cells (hMSC) within 48 h after printing. Furthermore, no difference in the proliferation behavior of printed cell lines and stem cells was detected [12, 14]. Since the laser pulse energy induces high temperature in the absorption layer, potential cell damage was also investigated via immunocytochemical studies, observing no increased expression of heat shock proteins by printed cells [3, 12].

Since stem cell differentiation can be induced by mechanical forces, the effect of laser-printing on the phenotype [14] and the differentiation potential [3, 16] of mesenchymal stem cells was studied. It was shown that phenotype and differentiation behavior were not influenced. So far, all studies consistently indicate that the by laser printing procedure with suitable parameters does not affect the cells.

Different printed 3D cell constructs are presented here, a mono-cellular 3D stem cell graft, a multicellular 3D skin equivalent, and a defined 3D spot array for microscopically observation of cell-cell and cell-environment interactions.

Printing cell droplet arrays for cell-cell and cell-environment interaction studies

Recent studies indicate [17] that local cell density, cellular spacing, cell-cell communication, and binding of cells to their 3D environment affect or control cell behavior and tissue functionality. Cellular microarrays were developed [18] to reproducibly study effects of drugs, proteins, growth factors, and biomaterials.

Gruene et al. [19] applied LaBP for the assembly of multi-cellular 3D arrays layer-by-layer, consisting of discrete droplets of human adipose-derived stem cells (ASCs) or endothelial colony-forming cells (ECFCs). Thereby, any cell-cell ratio, cell quantity, cell-type combination, and spot spacing can be realized within such an array; and the height of the 3D array is freely scalable. A natural sol consisting of a fibrinogen and hyaluronic acid served as the cell carrier and environmental matrix. First, a layer of fibrin is produced on the collector slide by blade-coating fibrinogen and subsequently crosslinking with thrombin. Second, different cell types are printed on top of the first fibrin layer at a controlled cell spot-spacing by LaBP. Third, a second fibrin layer is blade-coated using the same procedure. Then, the second and third steps can be repeated several times to produce true 3D cell arrays. Alternatively, the fibrin-based environment can be replaced by any other hydrogel.

ASCs and ECFCs were chosen for investigating vascular network formation, since these cell types represent suitable cell sources for therapeutic re-vascularization of ischemic tissues and can support vessel formation in engineered tissue constructs [20].

In Figure 2, separate printed spots of ASCs and ECFCs with a planar spot distribution are depicted. Cell-cell interactions in vascular endothelial growth factor-free (VEGF-free) medium were observed for 10 days. Additionally, monoculture 3D cell arrays consisting of ASCs or ECFCs were generated and kept in VEGF-free culture medium as control. As shown in Figure 2C, the ASCs (+) migrated towards the ECFCs (o) and contacted them on day 3 [19]. Until then the ECFCs showed negligible activity. After the cell-cell contact on day 3, the ECFCs’ activity strongly increased. They began to form vascular-like networks, which grew out towards the ASC spots and formed big branches (see Figure 2D). These networks remained stable for 2 weeks under culture conditions and were not observable either in the ASC control or the ECFC control. The observed migration of ASCs towards ECFCs may be due to a gradient of platelet-derived growth factor (PDGF). The subtype PDGF-BB is expressed in large amounts by ECFCs and is well known for the stimulation of ASC proliferation and migration [21].

(A–D) Visualization of cell-cell interactions by 3D cell arrays in mono- and co-cultures, cultivated for 5 days under VEGF-free conditions. A circle indicated the printed ECFC spot and a cross the printed ASC spot. Interactions of ASCs and ECFCs (C, D) in comparison to separated arrays of ECFCs (B) and ASCs (A). A vascular-like network formation occurs after 5 days in the co-culture (D). Distance between spots with the same cell type is 800 μm. Scale bar=800 μm (A, B).
Figure 2

(A–D) Visualization of cell-cell interactions by 3D cell arrays in mono- and co-cultures, cultivated for 5 days under VEGF-free conditions. A circle indicated the printed ECFC spot and a cross the printed ASC spot. Interactions of ASCs and ECFCs (C, D) in comparison to separated arrays of ECFCs (B) and ASCs (A). A vascular-like network formation occurs after 5 days in the co-culture (D).

Distance between spots with the same cell type is 800 μm. Scale bar=800 μm (A, B).

Printing stem cell grafts

In regenerative medicine, stem cells are of widespread interest due to their capability of self-renewal and differentiation, which is regulated by their 3D microenvironment. Within this so-called stem cell niche, cell density, cell-cell contacts, cell-matrix adhesion, and the exchange of growth factors and oxygen are important [17]. Therefore, a key issue in stem cell biology is the fabrication of niche-like environments as in vitro models.

Gruene et al. [3, 16] printed mesenchymal stem cells [MSCs, porcine bone-marrow derived (pMSCs) and human adipose derived (ASCs)] in monocellular 3D patterns (approximately 300 μm high) to study if these stem cell grafts can be differentiated within the printed pattern and if the printing process affects the stem cells differentiation potential towards different lineages (osteogenic, chondrogenic, and adipogenic). A mixture of alginate and ethylene-diamine-tetra-acetic (EDTA) blood plasma was applied as the sol for printing and cross-linked with calcium chloride to form a firm extracellular matrix. This material is compatible with the cells and enables the exchange of nutrients and soluble factors.

However, material elasticity and forces can influence stem cell differentiation [21] as well as the initial cell density. Especially, chondrogenesis requires a high cell density.

It was investigated quantitatively and qualitatively, if the laser printing procedure has an effect on stem cell behavior, since stem cells may undergo forces during the printing procedure that would not affect their proliferation ability, but might induce uncontrolled differentiation [22].

The quantitative assessments (not shown) demonstrated no significant difference in the alkaline phosphatase activity and calcium accumulation from printed and non-printed control MSCs under osteogenic differentiation conditions. Similar, the accumulation of lipids under adipogenic culture conditions and of sGAG (sulfated glycosaminoglycans) for chondrogenesis, under chondrogenic culture conditions did not differ significantly between printed and non-printed control cells. Thus, it could be demonstrated that the differentiation potential of MSCs into osteogenic, adipogenic, and chondrogenic lineages is not affected by the printing procedure.

Additionally, printed grid patterns (Figure 3) were successfully differentiated over several weeks into bone, fat, and cartilage tissue grafts [3, 16], indicating that LaBP is capable of printing cell densities high enough for the promotion of chondrogenesis. 3D scaffold-free tissue grafts were fabricated with LaBP keeping their predefined shape after several weeks in culture, even if removing the matrix material after 2 weeks in culture. These facts are major requirements for the successful fabrication of 3D stem cell constructs.

Osteogenic, adipogenic and chondrogenic differentiation of printed mesenchymal stem cells (MSC). (A–D) Printed 3D grid structures of MSC: directly after printing (A), after 25 days under osteogenic (B) and after 21 days under adipogenic (C), or chondrogenic (D) culture conditions: microscopic images with phase contrast (A2, B1, B2, C1, D1), life-dead-staining (A1; Calcein AM/Homodimer E1), Oil-Red-O staining (C2) and collagen type II immunofluorescence (D2). Accumulation of calcium phosphate for osteogenic differentiation (B), lipid vacuoles indicating adipogenic differentiation (C) and collagen type II (D2), specific for chondrogenesis, can be seen.
Figure 3

Osteogenic, adipogenic and chondrogenic differentiation of printed mesenchymal stem cells (MSC).

(A–D) Printed 3D grid structures of MSC: directly after printing (A), after 25 days under osteogenic (B) and after 21 days under adipogenic (C), or chondrogenic (D) culture conditions: microscopic images with phase contrast (A2, B1, B2, C1, D1), life-dead-staining (A1; Calcein AM/Homodimer E1), Oil-Red-O staining (C2) and collagen type II immunofluorescence (D2). Accumulation of calcium phosphate for osteogenic differentiation (B), lipid vacuoles indicating adipogenic differentiation (C) and collagen type II (D2), specific for chondrogenesis, can be seen.

Printing skin tissue

The major objective and challenge for cell printing techniques is the fabrication of functional tissue, mimicking the structure and characteristics of natural tissue. For most types of tissues, this includes several different cell types and the generation of a vascular network, to supply the cells by perfusion. So far, bioprinting is far away from reaching this aim.

To prove the tissue generation potential of LaBP, skin tissue equivalents were printed with 20 layers of fibroblasts and 20 layers of keratinocytes, both embedded in collagen type I (Figure 4A). This mimics the layered structure of natural skin with dermis and epidermis [23].

Fluorescence microscopic images of 3D printed fibroblasts and keratinocytes in collagen type I as a skin mimicking bi-layered construct; cryostat sections were prepared 10 days after printing, except (A, G). All scale bars are 50 μm. (A): A section through the laser printed structure, prepared directly after printing, with transduced fibroblasts (red) and keratinocytes (green). (B): The fibroblasts are stained in red (pan-reticular fibroblast), keratinocytes are stained in green (cytokeratin 14), and cell nuclei are stained in blue (Hoechst 33342). Especially the keratinocytes formed a compact cell organization. (C): Anti-laminin staining (green). Laminin is a major constituent of the basement membrane in skin. (D): Immunoperoxidase staining of cytokeratin 14 (upper brown layer) depicts keratinocytes in the bi-layered structure while fibroblasts can be seen in the light layer underneath. (E): Pan-Cadherin-staining depicts in red the adherens junctions consisting of cadherins, which are located between the membranes of neighboring cells. (F): Connexin-43 (Cx43) staining in green, connexins are the constituents of gap junctions; Cx43 is distributed in a scattered, punctate fashion, which is a sign for the formation of gap junctions. (G): Gap junction coupling visualized with Lucifer yellow (green) after scrape loading procedure, the keratinocytes, transfected with mCherry, are depicted in red. (B, C, E, F): All cell nuclei are stained with Hoechst 33342 (blue).
Figure 4

Fluorescence microscopic images of 3D printed fibroblasts and keratinocytes in collagen type I as a skin mimicking bi-layered construct; cryostat sections were prepared 10 days after printing, except (A, G). All scale bars are 50 μm. (A): A section through the laser printed structure, prepared directly after printing, with transduced fibroblasts (red) and keratinocytes (green). (B): The fibroblasts are stained in red (pan-reticular fibroblast), keratinocytes are stained in green (cytokeratin 14), and cell nuclei are stained in blue (Hoechst 33342). Especially the keratinocytes formed a compact cell organization. (C): Anti-laminin staining (green). Laminin is a major constituent of the basement membrane in skin. (D): Immunoperoxidase staining of cytokeratin 14 (upper brown layer) depicts keratinocytes in the bi-layered structure while fibroblasts can be seen in the light layer underneath. (E): Pan-Cadherin-staining depicts in red the adherens junctions consisting of cadherins, which are located between the membranes of neighboring cells. (F): Connexin-43 (Cx43) staining in green, connexins are the constituents of gap junctions; Cx43 is distributed in a scattered, punctate fashion, which is a sign for the formation of gap junctions. (G): Gap junction coupling visualized with Lucifer yellow (green) after scrape loading procedure, the keratinocytes, transfected with mCherry, are depicted in red. (B, C, E, F): All cell nuclei are stained with Hoechst 33342 (blue).

Two well-established cell lines, murine fibroblasts (NIH3T3) and human keratinocytes (HaCaT from adult human skin), were used, which were also combined in other studies. 3T3 fibroblasts secreting growth factors favorable for keratinocytes [24] and therefore, they are widely used in the cultivation of keratinocytes. The most abundant protein in skin, collagen, was applied as hydrogel.

Since tissue formation is determined by intercellular junctions [25], the existence of these junctions was investigated. Specific junctions can be found as cell-cell and cell-matrix connections in all kinds of tissue, abundantly in epithelium like the epidermis (consisting mainly of keratinocytes).

Intercellular adherens junctions [26] are fundamental for tissue morphogenesis and cohesion, composed mainly of cadherins (calcium-dependent adherent proteins). The extensive formation of intercellular adherens junctions between printed keratinocytes as well as a minor formation between fibroblasts as expected can be seen in Figure 4E.

Gap junctions [27] allow intercellular communication by chemical signals passing through these cell-cell-channels. They consist of connexins and are known to have a fundamental role in differentiation, cell cycle progression, and cell survival.

Immunostaining (Figure 4F) of Connexin 43 (Cx43), the main connexin in human skin, showed its expression in all cells, localized within the cell membrane. Cx43 is distributed in a scattered, punctate fashion, which is a sign for the formation of gap junctions [28]. The functionality of cell-cell communication via gap junction coupling was assessed with a dye-transfer, so-called scrape loading method [29] in vital 3D cell constructs. Therefore, the dye’s diffusion distance over channel-connected neighboring cells, correlating with gap junction coupling, was analyzed (Figure 4G). The dye went through up to ten successive cells within 5 min, which demonstrates a good gap junction coupling.

Thus, the printed skin grafts mimic tissue-specific functions with respect to adherens and gap junctions [23].

Furthermore, the formation of a basement membrane between keratinocytes and fibroblasts could be observed (Figure 4C), as it exists between epidermis and dermis in natural skin.

These skin constructs were also tested in vivo in nude mice. They were placed into full-thickness skin wounds and were fully connected to the surrounding tissue after 11 days. The printed skin constructs already exhibit a similar basic structure than natural skin [30], albeit it lacks several skin components like hair, sweat glands or blood vessels. A multi-layered epidermis was formed by the printed keratinocytes with beginning differentiation and stratum corneum. Blood vessels have been found to grow from the wound bed and the wound edges into the printed cells [30].

Conclusions

Laser-assisted bioprinting (LaBP) is a promising micro-fabrication technique applied for arranging vital cells in pre-defined two and three-dimensional patterns. With many different cell types (cell lines, primary cells, and stem cells), it was proven that this technique (with suitable parameters) does not harm the printed cells or influence the differentiation potential of stem cells. Cell quantities ranging from single to tens of cells per droplet, embedded in hydrogel precursors with a wide range of rheological properties, have been printed with micrometer resolution. Besides cells, also growth factors or biological agents can be printed.

Different applications, demonstrating the potential of LaBP, are presented. With LaBP, 3D scaffold-free stem cell grafts were fabricated, cells were arranged in 3D spot arrays for cell-cell and cell-environment interaction studies, and the 3D arrangement of vital cells by laser-printing as multicellular grafts analogous to native skin archetype demonstrated tissue formation by printed cells.

Reproducibly fabricated human tissue equivalents and defined 3D cell patterns offer new avenues in biomedical research. They can serve as 3D environments for studying cell behavior. With further integrated cell types, like endothelial cells for vascularization or dendritic cells for immune reactions, the skin tissue equivalents can be used for testing pharmaceutical or cosmetic agents to reduce animal testing; the use of human cells and the reproducibility in fabrication is thereby an advantage compared to laboratory animals. By printing also other tissue types, defined body-on-a-chip systems might be generated.

Besides the development of printing devices, the engineering and adaption of hydrogels as bio-inks for printing is of vital importance for further progress towards printed tissue. Thereby, hydrogels serve as the extra-cellular matrix of the printed construct, but they (or polymer solutions) also might be printed as sacrificial structures, which are dissolved after printing to generate hollow tubes or cavities inside the printed construct, especially for vascularization [31].

Mostly, natural hydrogels like alginate, hyaluronic acid, fibrin, collagen, gelatin, or agarose are used, but also synthetic hydrogels or polymers like poly(ethylene glycol) (PEG) are under investigation with different printing techniques, primarily with extrusion systems [32]. Thereby, the adaption of the gelation and biodegrading dynamics of these hydrogels to the printing technique and the biomedical application [33] is one focus. Also combinations of thermoplastic polymers and hydrogels are used; Catros et al. [34] combined thermoplastic electrospun polycaprolacton with printed alginate.

Furthermore, complex hydrogels are generated, which for example include the ability to deliver bioactive factors on a predefined time-scale. Thus, growth factors could be released to guide the tissue maturation after printing [35].

The development of reproducible, controllable, and well-defined 3D cell models is a key challenge for future progress in tissue engineering. Therefore, the ability to precisely position different cells in complex 3D patterns is of essential importance.

Acknowledgments

The studies described here have been supported by Deutsche Forschungsgemeinschaft, SFB TransRegio 37, REBIRTH Cluster of Excellence (Exc62/1), and by Land Niedersachsen and Volkswagenstiftung in the Biofabrication for NIFE project.

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About the article

Corresponding author: Lothar Koch, Laser Zentrum Hannover e.V. Nanotechnology Department, Hollerithallee 8, D-30419 Hannover, Germany, Phone: +49 511 2788-256, Fax: +49 511 2788-100, E-mail: , Internet: www.lzh.de


Received: 2014-05-06

Accepted: 2014-10-15

Published Online: 2014-11-12

Published in Print: 2014-12-01


Citation Information: BioNanoMaterials, Volume 15, Issue 3-4, Pages 71–78, ISSN (Online) 2193-066X, ISSN (Print) 2193-0651, DOI: https://doi.org/10.1515/bnm-2014-0005.

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