Name: viability of bioprinted cellular constructs using a three dispenser cartesian printer
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Description: Watch this Scientific Journal Video about Viability of Bioprinted Cellular Constructs Using a Three Dispenser Cartesian Printer at JoVE.com

This work was supported by Government Support under Grant No. EPS-0903795 awarded by the National Science Foundation, NIH NIDCR R01-DE019355 (MJY PI), and Grant 8P20 GM103444 (YM PI).

1. Preparation of Gelatin Containing Substrate for Three-Dimensional Bioprinting of Alginate Hydrogels
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	<li>Prepare the calcium/gelatin substrate following the calcium/gelatin substrate method described by Pataky et al11 to avoid reduced viability associated with high content. The calcium/gelatin substrate method is listed below.
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		<li>Combine calcium chloride dehydrate (1.5 wt%), sodium chloride (0.9 wt%), and porcine gelatin (2 wt%) in distilled water and boil for 2 min&#160;to create a...

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The primary focus of tissue engineering is to bridge the gap between organ shortages and transplantation needs by developing biological substitutes capable of restoring, maintaining, or improving native tissue functio. This has led to the direct fabrication of scaffolds with a complex, anatomically correct external geometry, and precise control over the internal geometr. Three-dimensional bioprinting is a methodology used for generating three-dimensional constructs of various sizes and shapes from a digital model using a...

Tissue engineering uses the principles of biology and engineering in the development of functional substitutes to maintain, restore, or enhance native tissue and . The capability of generating three-dimensional biomimetic constructs on demand would facilitate scientific and technological advances in tissue engineering as well as in cell-based sensors, drug/toxicity screening, tissue or tumor models, and other . The three-dimensional organization of tissue-engineered constructs is a fundamental component of the fabrication method because it must closely mimic the highly organized interaction of cells and extracellular matrix in native tissue.
Biodegradable and shape-forming three-dimensional scaffolds are critical factors in generating novel tissue constructs because cells migrate to form a two-dimensional layer of cells, but lack the ability to grow in favored three-dimensional . The scaffold serves as a temporary foundation for cell attachment and proliferation, so it must be constructed from materials with controllable porosity and biodegradability, and sufficient mechanical integrit. The scaffold materials should not be cytotoxic or create an adverse response from the host. Hydrogels have been commonly used in tissue engineering techniques, and due to their hydrophilicity, hydrogels permit fluid and gas exchange throughout the structur. By combining different hydrogels, the synthesized hydrogel&#8217;s properties are modifiable to meet distinct application requirement.
The conventional tissue engineering approach involves the creation of acellular porous sacrificial scaffolds that are seeded with cells post-fabricatio. Many techniques have been employed, such as fiber bonding, solvent casting, and melt molding, but proved to be minimally successful for tissue engineering applications. Fiber bonding methods allow fibers to be aligned in specific shapes, but they are only capable of producing very thin scaffold. Solvent casting methods produced highly porous constructs, however the largest produced membrane was only 3-mm thic. Therefore, creating three-dimensional constructs is not feasible using these techniques. Melt molding techniques proved successful in producing three-dimensional scaffolds, but such high temperatures are required that biological materials cannot be incorporated during the production proces. Scaffolds seeded post-fabrication are limited in their ability to meet the requirements of tissue engineering to produce three-dimensional scaffolds with pre-defined or controllable microstructures and . Another major issue with solid scaffold seeding technologies is the deficiency of vascularization and poor mechanical .
Bioprinting has since been extended to three dimensions through the use of nontoxic, biodegradable, thermo-reversible gels to overcome the disadvantages of conventional . A few of the solid freeform fabrication techniques currently being employed are laser-assisted bioprinting and inkjet printing. Laser-assisted bioprinting techniques use a pulsed laser source, a target plate, and a receiving substrate to generate three-dimensional . However, this technique is limited due to low throughput, low cell viability, and can only produce limited arrangements of fabricated structures because only photocrosslinkable prepolymers can be used to form a crosslinked hydrogel . Inkjet printing was developed as a non-contact methodology that reproduces digital image data on a substrate by depositing picoliter ink . However, inkjet printing does not produce a high-resolution construct, constructs experience rapid protein denaturation, and many of the cells are lysed during the deposition .
Currently, new additive manufacturing bioprinting methods have been developed. In these systems cells, proteins, growth factors, and biomimetic hydrogels are typically integrated into matrix materials during the fabrication process and concurrently deposited using computer-controlled actuators to generate three-dimensional scaffold-based cell-laden constructs that closely mimic the microarchitecture of native . The cell-laden hydrogels constitute the bioink, which can be heterogeneous, consisting of multiple cell types, or homogeneous. Additive manufacturing systems deposit bioink drop-by-drop or layer-by-layer via disposable syringes and tips onto a computer-controlled stage capable of moving in the x, y, and z directions. Through computer software, the architecture of printed scaffolds can be easily manipulated depending on requirements of the application. Unlike conventional techniques, three-dimensional medical technologies (magnetic resonance imaging, computer tomography) can be incorporated into the designs, generating patient-specific construct. These methods also allow the possibility of producing vascularized replacements because constructs are produced with a higher local cell density, allowing cell-cell interactions and improving the likelihood of post-implantation surviva.
The Palmetto Printer is a custom built three-dimensional multi-dispenser system that uses programmable robotic manufacturing methods to generate three-dimensional heterogeneous tissue constructs (Figure 1). It allows the use of a plurality of materials in unique combinations to produce heterogeneous structures. The initialization of the bioprinter is one of the most important steps in bioprinting because it allows you to set a variety of parameters to optimize the printability of the bioprinted constructs.
The bioprinter comprises a batch type process with startup, operation and shutdown sequences controlled by a programmable logic controller (PLC), which the user operates through an interactive touch screen control panel (Figure 1,&#160;A). To prevent contamination of biological materials the bioprinter is enclosed in a positively-pressured poly(methyl methacrylate) (PMMA) chamber with a high-efficiency particulate arrestance (HEPA)-filtered air circulation system (Figure 1, B,C). The interior of the printer can be sterilized using the built-in ultraviolet light sources (Figure 1, D). The central component of the bioprinter is a fully programmable positioning robot that can reproducibly place a dispenser tip with an accuracy of 10 micrometers (Figure 1, E). There are three dispensers, which are able to deposit volumes as small as 230 nl using a rotary screw (Figure 1, F). They are independently programmable using separate computers that govern printing parameters for each dispenser (Figure 1, G). Rotary-screw dispensing utilizes the rotation of a motor-driven screw to move bioink down a syringe and out of the syringe tip. These dispensers are mounted onto a pneumatically controlled Tool Nest (Figure 2A, B), allowing the robot to switch dispenser mounted onto the Z-axis robotic arm under programmed control (Figure 1, H).
The XYZ robot receives printing instructions from a computer running design software (Figure 1, I). Each program contains dispensing locations, calibration routines, and dispenser-changing protocols. The design of generated constructs primarily consists of the XYZ coordinates where each dispenser will deposit material. The bioprinter comprises two optical light sensors (Figure 2C) that determine the XYZ coordinates of the syringe tip end. These sensors send coordinate information to the robot, which uses these to calculate positions of the dispenser tip ends. There is an additional displacement laser (Figure 2D) that projects a 633 nm diode red laser beam of spot size 30 x 100 micrometers to measure distance with an accuracy of 0.1 micrometers. When the beam is highly focused the robot determines the Z distance of the printing surface. This measurement, and the optical light sensors measurement of the tip end in Z, allows calculation of accurate Z coordinates used to place the dispenser tip in relation to the printing surface. The dispenser tips move laterally and vertically through the X-axis oriented optical light sensor to find the Y and Z centers, and laterally through a Y-axis sensor to find the center of the X-axis. The printing surface is mapped using the formula for a flat plane in xyz space: ax + by +cz = d to determine where the surface is relative to the position of the dispensing tip end. The printer stage (Figure 1, J) holds a sample Petri dish up to 80 mm in diameter and uses a recirculating water bath to maintain the set temperature (Figure 1, K). Stage temperature can be set within a range of -20 and remains stable within . There is a USB camera mounted onto the robot Z-arm to provide a magnified view of the dispensing tip during the printing process (Figure 1, L). There is a second camera mounted towards the top of the chamber interior that provides a complete view of the bioprinter during the printing process (Figure 1, L).
A computer-aided design drawing software determines the deposition pattern and permits the user to generate incrementally spaced droplets and complex structures (Figure 3). Three-dimensional pathways can be manually coded into the printer-compatible design software or imported from a separate computer-aided design drawing software (Figure 4, Table 1). The printer-compatible software allows variations of printing parameters such as the deposition method (single droplet deposition or continuous pathway deposition), three-dimensional geometry of the pathways, deposition rate, distance between the syringe tip end and substrate printing surface, the amount of time to deposit an individual drop, and the height and speed the syringe is lifted between deposition of the drops. Each program contains XYZ dispensing locations, tip calibration routines, and dispenser-changing protocols to provide a sterile environment, without operator intervention, during printing. The programmable logic controller (PLC) of the robot receives instructions from the computer running the design software and controls the timing of events from the external controllers (e.g.,&#160;the dispensers). To do this, the PLC uses a looping mechanism to control the dispensers, robotic positioning device, and environmental factors.
Three-dimensional direct-write bioprinting utilizing a rotary-screw, liquid-dispensing system allows the process of depositing cells to be more efficient, accurate, and easier than previous methods. This study shows the custom built bioprinter is capable of generating cell-laden hydrogel constructs with high cell viability.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Positioning Robot (JR2000 XYZ)</td>
			<td>Janome&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dispensers: SDAV Linear Drive SmartDispensers</td>
			<td>Fishman Corporation</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Optical Light Sensors:&#160;</td>
			<td>Keyensce</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Displacement Laser: OD Mini</td>
			<td>SICK</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Recirculating Water Bath: Polystat</td>
			<td>Cole-Parmer</td>
			<td>EW-12122-02</td>
			<td></td>
		</tr>
		<tr>
			<td>USB Cameras: Dino-Lite Premier 5MP</td>
			<td>AnMo Electrionics/YSC Technologies</td>
			<td>AD7013MT</td>
			<td></td>
		</tr>
		<tr>
			<td>Printer-Compatible Computer Design Software: JR-C Points</td>
			<td>Janome</td>
			<td></td>
			<td>Comes with purchase of Janome Robot</td>
		</tr>
		<tr>
			<td>Computer-Aided Design Drawing Software: Visual PathBuilder</td>
			<td>RatioServ</td>
			<td></td>
			<td>Can be downloaded at: www.ratioserv.com/index.php/downloads</td>
		</tr>
		<tr>
			<td>Printer 3 cc Syringes:&#160;</td>
			<td>Fishman Corporation</td>
			<td>122051</td>
			<td></td>
		</tr>
		<tr>
			<td>22 G Dispenser Tips</td>
			<td>Fishman Corporation</td>
			<td>Z520122&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium Chloride Dihydrate</td>
			<td>Sigma-Aldrich</td>
			<td>10035-04-8</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Sigma-Aldrich</td>
			<td>7647-14-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Porcine Gelatin</td>
			<td>Sigma-Aldrich</td>
			<td>9000-70-8</td>
			<td></td>
		</tr>
		<tr>
			<td>Titanium Dioxide</td>
			<td>Sigma-Aldrich</td>
			<td>13462-67-7</td>
			<td></td>
		</tr>
		<tr>
			<td>Protanal LF 20/40 Alginate (Sodium Alginate)</td>
			<td>FMC BioPolymer</td>
			<td>9005-38-3</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrochloric Acid</td>
			<td>Sigma-Aldrich</td>
			<td>7647-01-0</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethylene Glycol</td>
			<td>Mallinckrodt Baker, Inc</td>
			<td>9300-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Periodate</td>
			<td>Sigma-Aldrich</td>
			<td>7790-28-5</td>
			<td></td>
		</tr>
		<tr>
			<td>hADSC</td>
			<td>Lonza</td>
			<td>PT-5006</td>
			<td>Store in vials in liquid nitrogen until use.</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle&#39;s Medium</td>
			<td>Gibco Life Technologies</td>
			<td>11965-092</td>
			<td>Warm in 37&#176;C water before use.</td>
		</tr>
		<tr>
			<td>Trypsin/EDTA</td>
			<td>Lonza</td>
			<td>CC-5012</td>
			<td>Warm in 37&#176;C water before use.</td>
		</tr>
		<tr>
			<td>Calcein AM</td>
			<td>Gibco Life Technologies</td>
			<td>C3100MP</td>
			<td>Store in the dark at -80&#176;C until use.</td>
		</tr>
		<tr>
			<td>Live/Dead Mammalian Viability Assay Kit</td>
			<td>Invitrogen Life Technologies</td>
			<td>L-3224</td>
			<td>Store in the dark at -80&#176;C until use.</td>
		</tr>
		<tr>
			<td>MES Hydrate</td>
			<td>Sigma-Aldrich</td>
			<td>M2933</td>
			<td></td>
		</tr>
		<tr>
			<td>N-Hydroxysuccinimide</td>
			<td>Sigma-Aldrich</td>
			<td>130672</td>
			<td></td>
		</tr>
		<tr>
			<td>1-ethyl-(dimethylaminopropyl) carbodiimide (EDC)</td>
			<td>Sigma-Aldrich</td>
			<td>E1769&#160;</td>
			<td>10 G</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Phosphate-Buffered Saline, +Calcium, +Magnesium</td>
			<td>Life Technologies</td>
			<td>14040133</td>
			<td>Warm in 37&#176;C water before use.</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Phosphate-Buffered Saline, -Calcium, -Magnesium</td>
			<td>Life Technologies</td>
			<td>14190144</td>
			<td>Warm in 37&#176;C water before use.</td>
		</tr>
		<tr>
			<td>RGD Peptides</td>
			<td>International Peptides</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 546 Phalloidin Stain</td>
			<td>Invitrogen Life Technologies</td>
			<td>A22283</td>
			<td>Store at -20&#176;C until use</td>
		</tr>
		<tr>
			<td>(4&#8217;, 6-Diamidino-2-Phenylindole, Dihydrochloride) (DAPI) Stain</td>
			<td>Life Technologies</td>
			<td>R37606</td>
			<td>Store at -20&#176;C until use</td>
		</tr>
	</tbody>
</table>

viability of bioprinted cellular constructs using a three dispenser cartesian printer

Tissue engineering has centralized its focus on the construction of replacements for non-functional or damaged tissue. The utilization of three-dimensional bioprinting in tissue engineering has generated new methods for the printing of cells and matrix to fabricate biomimetic tissue constructs. The solid freeform fabrication (SFF) method developed for three-dimensional bioprinting uses an additive manufacturing approach by depositing droplets of cells and hydrogels in a layer-by-layer fashion. Bioprinting fabrication is dependent on the specific placement of biological materials into three-dimensional architectures, and the printed constructs should closely mimic the complex organization of cells and extracellular matrices in native tissue. This paper highlights the use of the Palmetto Printer, a Cartesian bioprinter, as well as the process of producing spatially organized, viable constructs while simultaneously allowing control of environmental factors. This methodology utilizes computer-aided design and computer-aided manufacturing to produce these specific and complex geometries. Finally, this approach allows for the reproducible production of fabricated constructs optimized by controllable printing parameters.

The results demonstrate the bioprinter is capable of depositing cell-laden hydrogels in specific three-dimensional locations accurately and consistently using computer-aided software. These softwares determine the placement of each droplet and control many of the parameters for dispensing (Figure 3,4). The repeatability of the bioprinter to appropriately deposit biomaterials is fundamental to its success in tissue engineering applications.
Cell viability, one of the requiremen...

Watch this Scientific Journal Video about Viability of Bioprinted Cellular Constructs Using a Three Dispenser Cartesian Printer at JoVE.com

Viability of Bioprinted Cellular Constructs Using a Three Dispenser Cartesian Printer

A Cartesian bioprinter was designed and fabricated to allow multi-material deposition in precise, reproducible geometries, while also allowing control of environmental factors. Utilizing the three-dimensional bioprinter, complex and viable constructs may be printed and easily reproduced.

Tissue engineering has centralized its focus on the construction of replacements for non-functional or damaged tissue. The utilization of ...

viability-bioprinted-cellular-constructs-using-three-dispenser

Research

JoVE Journal

Bioengineering

The Three-Dimensional Human Skin Reconstruct Model: a Tool to Study Normal  Skin and Melanoma Progression

Most in vitro studies in experimental skin biology have been done in 2-dimensional (2D) monocultures, while accumulating evidence suggests that cells behave differently when they are grown within a 3D extra-cellular matrix and also interact with other cells (1-5). Mouse models have been broadly utilized to study tissue morphogenesis in vivo. However mouse and human skin have significant differences in cellular architecture and physiology, which makes it difficult to extrapolate mouse studies to humans. Since melanocytes in mouse skin are mostly localized in hair follicles, they have distinct biological properties from those of humans, which locate primarily at the basal layer of the epidermis. The recent development of 3D human skin reconstruct models has enabled the field to investigate cell-matrix and cell-cell interactions between different cell types. The reconstructs consist of a "dermis" with fibroblasts embedded in a collagen I matrix, an "epidermis", which is comprised of stratified, differentiated keratinocytes and a functional basement membrane, which separates epidermis from dermis. Collagen provides scaffolding, nutrient delivery, and potential for cell-to-cell interaction. The 3D skin models incorporating melanocytic cells recapitulate natural features of melanocyte homeostasis and melanoma progression in human skin. As in vivo, melanocytes in reconstructed skin are localized at the basement membrane interspersed with basal layer keratinocytes. Melanoma cells exhibit the same characteristics reflecting the original tumor stage (RGP, VGP and metastatic melanoma cells) in vivo. Recently, dermal stem cells have been identified in the human dermis (6). These multi-potent stem cells can migrate to the epidermis and differentiate to melanocytes.

In this report, we describe the three-dimensional skin reconstruct model which mimics human skin in architecture and composition. Melanocyte physiology, melanoma progression and the fate of dermal stem cells have been investigated using the skin reconstruct model. The model is also useful as a preclinical tool for drug assessment.

Most in vitro studies in experimental skin biology have been done in 2-dimensional (2D) monocultures, while accumulating evidence suggests that cells ...

Creating Transient Cell Membrane Pores Using a Standard Inkjet Printer

Bioprinting has a wide range of applications and significance, including tissue engineering, direct cell application therapies, and biosensor microfabrication.1-10 Recently, thermal inkjet printing has also been used for gene transfection.8,9 The thermal inkjet printing process was shown to temporarily disrupt the cell membranes without affecting cell viability. The transient pores in the membrane can be used to introduce molecules, which would otherwise be too large to pass through the membrane, into the cell cytoplasm.8,9,11
The application being demonstrated here is the use of thermal inkjet printing for the incorporation of fluorescently labeled g-actin monomers into cells. The advantage of using thermal ink-jet printing to inject molecules into cells is that the technique is relatively benign to cells.8, 12 Cell viability after printing has been shown to be similar to standard cell plating methods1,8. In addition, inkjet printing can process thousands of cells in minutes, which is much faster than manual microinjection. The pores created by printing have been shown to close within about two hours. However, there is a limit to the size of the pore created (~10 nm) with this printing technique, which limits the technique to injecting cells with small proteins and/or particles. 8,9,11
A standard HP DeskJet 500 printer was modified to allow for cell printing.3, 5, 8 The cover of the printer was removed and the paper feed mechanism was bypassed using a mechanical lever. A stage was created to allow for placement of microscope slides and coverslips directly under the print head. Ink cartridges were opened, the ink was removed and they were cleaned prior to use with cells. The printing pattern was created using standard drawing software, which then controlled the printer through a simple print command. 3T3 fibroblasts were grown to confluence, trypsinized, and then resuspended into phosphate buffered saline with soluble fluorescently labeled g-actin monomers. The cell suspension was pipetted into the ink cartridge and lines of cells were printed onto glass microscope cover slips. The live cells were imaged using fluorescence microscopy and actin was found throughout the cytoplasm. Incorporation of fluorescent actin into the cell allows for imaging of short-time cytoskeletal dynamics and is useful for a wide range of applications.13-15

A description of the methods used to convert an HP DeskJet 500 printer into a bioprinter. The printer is capable of processing living cells, which causes transient pores in the membrane. These pores can be utilized to incorporate small molecules, including fluorescent G-actin, into the printed cells.

Bioprinting has a wide range of applications and significance, including tissue engineering, direct cell application therapies, and biosensor ...

In Vivo Quantitative Assessment of Myocardial Structure, Function, Perfusion and Viability Using Cardiac Micro-computed Tomography

The use of Micro-Computed Tomography (MicroCT) for in vivo studies of small animals as models of human disease has risen tremendously due to the fact that MicroCT provides quantitative high-resolution three-dimensional (3D) anatomical data non-destructively and longitudinally. Most importantly, with the development of a novel preclinical iodinated contrast agent called eXIA160, functional and metabolic assessment of the heart became possible. However, prior to the advent of commercial MicroCT scanners equipped with X-ray flat-panel detector technology and easy-to-use cardio-respiratory gating, preclinical studies of cardiovascular disease (CVD) in small animals required a MicroCT technologist with advanced skills, and thus were impractical for widespread implementation. The goal of this work is to provide a practical guide to the use of the high-speed Quantum FX MicroCT system for comprehensive determination of myocardial global and regional function along with assessment of myocardial perfusion, metabolism and viability in healthy mice and in a cardiac ischemia mouse model induced by permanent occlusion of the left anterior descending coronary artery (LAD).

In Vivo Quantitative Assessment of Myocardial Structure, Function, Perfusion and Viability Using Cardiac Micro-computed Tomography

This method outlines the use of Quantum Micro-Computed Tomography (MicroCT) to assess cardiac morphology, function, perfusion, metabolism and viability with iodinated contrast agent in mice with experimentally-induced myocardial ischemia. The technique can be applied for non-destructive high-throughput longitudinal in vivo imaging of various animal models of human heart disease.

The use of Micro-Computed Tomography (MicroCT) for in vivo studies of small animals as models of human disease has risen tremendously due to the fact that ...

Bioprinting Cellularized Constructs Using a Tissue-specific Hydrogel Bioink

Bioprinting has emerged as a versatile biofabrication approach for creating tissue engineered organ constructs. These constructs have potential use as organ replacements for implantation in patients, and also, when created on a smaller size scale as model "organoids" that can be used in in vitro systems for drug and toxicology screening.
Despite development of a wide variety of bioprinting devices, application of bioprinting technology can be limited by the availability of materials that both expedite bioprinting procedures and support cell viability and function by providing tissue-specific cues. Here we describe a versatile hyaluronic acid (HA) and gelatin-based hydrogel system comprised of a multi-crosslinker, 2-stage crosslinking protocol, which can provide tissue specific biochemical signals and mimic the mechanical properties of in vivo tissues. 
Biochemical factors are provided by incorporating tissue-derived extracellular matrix materials, which include potent growth factors. Tissue mechanical properties are controlled combinations of PEG-based crosslinkers with varying molecular weights, geometries (linear or multi-arm), and functional groups to yield extrudable bioinks and final construct shear stiffness values over a wide range (100 Pa to 20 kPa). Using these parameters, hydrogel bioinks were used to bioprint primary liver spheroids in a liver-specific bioink to create in vitro liver constructs with high cell viability and measurable functional albumin and urea output. This methodology provides a general framework that can be adapted for future customization of hydrogels for biofabrication of a wide range of tissue construct types.

We describe a set of protocols that together provide a tissue-mimicking hydrogel bioink with which functional and viable 3-D tissue constructs can be bioprinted for use in in vitro screening applications.

Bioprinting has emerged as a versatile biofabrication approach for creating tissue engineered organ constructs. These constructs have potential use as ...

Layered Alginate Constructs: A Platform for Co-culture of Heterogeneous Cell Populations

Many load bearing tissues possess structurally and functionally distinct regions, typically accompanied by different cell phenotypes with differential mechanosensing characteristics. Engineering and analysis of these tissue types remain a challenge. Layered hydrogel constructs provide an opportunity for investigating the interactions among multiple cell populations within single constructs. Alginate hydrogels are both biocompatible and allow for easy isolation of cells after experimentation. Here, we describe a method for the development of small sized dual layered alginate hydrogel discs. This process maintains high cell viability of human mesenchymal stem cells during the formation process and these layered discs can withstand unconfined cyclic compression, commonly used for stimulation of hMSCs undergoing chondrogenesis. These layered constructs can potentially be scaled up to include additional levels, and also be used to segregate cell populations initially after layering. This dual layer alginate hydrogel culture platform can be used for many different applications including engineering and analysis of cells of load bearing tissues and co-cultures of other cell types.

Engineering and analysis of load bearing tissues with heterogeneous cell populations are still a challenge. Here, we describe a method for creating bi-layered alginate hydrogel discs as a platform for co-culture of diverse cell populations within one construct.

Many load bearing tissues possess structurally and functionally distinct regions, typically accompanied by different cell phenotypes with differential ...

Imaging Cell Viability on Non-transparent Scaffolds &#8212; Using the Example of a Novel Knitted Titanium Implant

Intervertebral disc degeneration and disc herniation is one of the major causes of lower back pain. Depletion of extracellular matrix, culminating in nucleus pulposus (NP) extrusion leads to intervertebral disc destruction. Currently available surgical treatments reduce the pain but do not restore the mechanical functionality of the spine. In order to preserve mechanical features of the spine, total disc or nucleus replacement thus became a wide interest. However, this arthroplasty era is still in an immature state, since none of the existing products have been clinically evaluated.
This study intends to test the biocompatibility of a novel nucleus implant made of knitted titanium wires. Despite all mechanical advantages, the material has its limits for conventional optical analysis as the resulting implant is non-transparent. Here we present a strategy that describes in vitro visualization, tracking and viability testing of osteochondro-progenitor cells on the scaffold. This protocol can be used to visualize the efficiency of the cleaning protocol as well as to investigate the biocompatibility of these and other non-transparent scaffolds. Furthermore, this protocol can be used to show adherence pattern of cells as well as cell viability and proliferation rates on/in the scaffold. This in vitro biocompatibility testing assay provides a propitious tool to analyze cell-material interaction in non-transparent and opaque scaffolds.

Imaging Cell Viability on Non-transparent Scaffolds &amp;#8212; Using the Example of a Novel Knitted Titanium Implant

Here we present a fluorophore based imaging technique to detect cell viability on a non-transparent titanium scaffold as well as to detect glimpses of the scaffold impurities. This protocol troubleshoots the drawback of imaging cell-cell or cell-metal interactions on non-transparent scaffolds.

Intervertebral disc degeneration and disc herniation is one of the major causes of lower back pain. Depletion of extracellular matrix, culminating in ...

Treatment of Ligament Constructs with Exercise-conditioned Serum: A Translational Tissue Engineering Model

In vitro experiments are essential to understand biological mechanisms; however, the gap between monolayer tissue culture and human physiology is large, and translation of findings is often poor. Thus, there is ample opportunity for alternative experimental approaches. Here we present an approach in which human cells are isolated from human anterior cruciate ligament tissue remnants, expanded in culture, and used to form engineered ligaments. Exercise alters the biochemical milieu in the blood such that the function of many tissues, organs and bodily processes are improved. In this experiment, ligament construct culture media was supplemented with experimental human serum that has been &#39;conditioned&#39; by exercise. Thus the intervention is more biologically relevant since an experimental tissue is exposed to the full endogenous biochemical milieu, including binding proteins and adjunct compounds that may be altered in tandem with the activity of an unknown agent of interest. After treatment, engineered ligaments can be analyzed for mechanical function, collagen content, morphology, and cellular biochemistry. Overall, there are four major advantages versus traditional monolayer culture and animal models, of the physiological model of ligament tissue that is presented here. First, ligament constructs are three-dimensional, allowing for mechanical properties (i.e., function) such as ultimate tensile stress, maximal tensile load, and modulus, to be quantified. Second, the enthesis, the interface between boney and sinew elements, can be examined in detail and within functional context. Third, preparing media with post-exercise serum allows for the effects of the exercise-induced biochemical milieu, which is responsible for the wide range of health benefits of exercise, to be investigated in an unbiased manner. Finally, this experimental model advances scientific research in a humane and ethical manner by replacing the use of animals, a core mandate of the National Institutes of Health, the Center for Disease Control, and the Food and Drug Administration.

We present a model of ligament tissue in which three-dimensional constructs are treated with the human exercise-conditioned serum and analyzed for collagen content, function, and cellular biochemistry.

In vitro experiments are essential to understand biological mechanisms; however, the gap between monolayer tissue culture and human physiology is large, ...

Fabrication of a Multiplexed Artificial Cellular MicroEnvironment Array

Cellular microenvironments consist of a variety of cues, such as growth factors, extracellular matrices, and intercellular interactions. These cues are well orchestrated and are crucial in regulating cell functions in a living system. Although a number of researchers have attempted to investigate the correlation between environmental factors and desired cellular functions, much remains unknown. This is largely due to the lack of a proper methodology to mimic such environmental cues in vitro, and simultaneously test different environmental cues on cells. Here, we report an integrated platform of microfluidic channels and a nanofiber array, followed by high-content single-cell analysis, to examine stem cell phenotypes altered by distinct environmental factors. To demonstrate the application of this platform, this study focuses on the phenotypes of self-renewing human pluripotent stem cells (hPSCs). Here, we present the preparation procedures for a nanofiber array and the microfluidic structure in the fabrication of a Multiplexed Artificial Cellular MicroEnvironment (MACME) array. Moreover, overall steps of the single-cell profiling, cell staining with multiple fluorescent markers, multiple fluorescence imaging, and statistical analyses, are described.

This article describes the detailed methodology to prepare a Multiplexed Artificial Cellular MicroEnvironment (MACME) array for high-throughput manipulation of physical and chemical cues mimicking in vivo cellular microenvironments and to identify the optimal cellular environment for human pluripotent stem cells (hPSCs) with single-cell profiling.

Cellular microenvironments consist of a variety of cues, such as growth factors, extracellular matrices, and intercellular interactions. These cues are ...

Calorespirometry: A Powerful, Noninvasive Approach to Investigate Cellular Energy Metabolism

Many cell lines used in basic biological and biomedical research maintain energy homeostasis through a combination of both aerobic and anaerobic respiration. However, the extent to which both pathways contribute to the landscape of cellular energy production is consistently overlooked. Transformed cells cultured in saturating levels of glucose often show a decreased dependency on oxidative phosphorylation for ATP production, which is compensated by an increase in substrate-level phosphorylation. This shift in metabolic poise allows cells to proliferate despite the presence of mitochondrial toxins. In neglecting the altered metabolic poise of transformed cells, results from a pharmaceutical screening may be misinterpreted since the potentially mitotoxic effects may not be detected using model cell lines cultured in the presence of high glucose concentrations. This protocol describes the pairing of two powerful techniques, respirometry and calorimetry, which allows for the quantitative and noninvasive assessment of both aerobic and anaerobic contributions to cellular ATP production. Both aerobic and anaerobic respirations generate heat, which can be monitored via calorimetry. Meanwhile, measuring the rate of oxygen consumption can assess the extent of aerobic respiration. When both heat dissipation and oxygen consumption are measured simultaneously, the calorespirometric ratio can be determined. The experimentally obtained value can then be compared to the theoretical oxycaloric equivalent and the extent of the anaerobic respiration can be judged. Thus, calorespirometry provides a unique method to analyze a wide range of biological questions, including drug development, microbial growth, and fundamental bioenergetics under both normoxic and hypoxic conditions.

This protocol describes calorespirometry, the direct and simultaneous measurement of both heat dissipation and respiration, which provides a noninvasive approach to assess energy metabolism. This technique is used to assess the contribution of both aerobic and anaerobic pathways to energy utilization by monitoring the total cellular energy flow.

Many cell lines used in basic biological and biomedical research maintain energy homeostasis through a combination of both aerobic and anaerobic ...

Design of an Open-Source, Low-Cost Bioink and Food Melt Extrusion 3D Printer

Three-dimensional (3D) printing is an increasingly popular manufacturing technique that allows highly complex objects to be fabricated with no retooling costs. This increasing popularity is partly driven by falling barriers to entry such as system set-up costs and ease of operation. The following protocol presents the design and construction of an Additive Manufacturing Melt Extrusion (ADDME) 3D printer for the fabrication of custom parts and components. ADDME has been designed with a combination of 3D-printed, laser-cut, and online-sourced components. The protocol is arranged into easy-to-follow sections, with detailed diagrams and parts lists under the headings of framing, y-axis and bed, x-axis, extrusion, electronics, and software. The performance of ADDME is evaluated through extrusion testing and 3D printing of complex objects using viscous cream, chocolate, and Pluronic F-127 (a model for bioinks). The results indicate that ADDME is a capable platform for the fabrication of materials and constructs for use in a wide range of industries. The combination of detailed diagrams and video content facilitates access to low-cost, easy-to-operate equipment for individuals interested in 3D printing of complex objects from a wide range of materials.

The aim of this work is to design and construct a reservoir-based melt extrusion three-dimensional printer made from open-source and low-cost components for applications in the biomedical and food printing industries.

Three-dimensional (3D) printing is an increasingly popular manufacturing technique that allows highly complex objects to be fabricated with no retooling ...