3D Bioprinting of Organs

Colin Gillespie

AP Biology

December 19, 2015

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Every day, an average of twenty two people die waiting for an organ that never comes

(HHS 2013). That is twenty two deaths too many, twenty two deaths that could have been

prevented if only there was an organ available. This problem can be prevented with 3D printing.

There are numerous methods of 3D printing, one common method is inkjetting, a process that is

conceptually similar to a normal printer (Xu et. al. 2014). The advantage of inkjet printing is its

scalability, limited waste, and its capability of printing with good resolution (Xu et. al. 2014).

Another method is direct-write, pen based bioprinters. These can use significantly more viscous

inks, which allows for the creation of more complex structures. In general, a 3D printer, through

movement of the printing nozzle, body, and/or surface, is able to print in all three dimensions. A

three dimension structure is built by printing a X-Y layer, then moving up the Z axis to print the

next layer. This process is like building a block structure- each new layer is dependent on the

layer below for a support. The ink used in a 3D bioprinter is a cell suspension in an alginate

solution (Christensen et. al. 2015, Yu et. al. 2013, Xu et. al. 2014, Williams et. al. 2013, Lee et.

al. 2015). In this paper, I reviewed recent research on the uses and creation of 3D printed organs.

I provide a review on recent efforts on improving the printing process, in addition to current

applications of 3D bioprinting.

Improving the Process

Inkjet bioprinting printing has two submethods, continuous flow, where the ink is

continuously leaving the printing head, and drop-on-demand (DOD) printing, where the printer

drops a limited amount of ink at a select place (Xu et. al. 2014). Of the two, DOD is favored due

to the simplicity in controlling droplet size, and the elimination of waste satellite droplets (Xu et.
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al. 2014). Xu et. al. investigated the droplet formation during DOD printing process, specifically

the effects of cell concentration on the formation of satellite droplets, droplet velocity, size, and

breakup time (Xu et. al. 2014). It was found that when cell concentration changes, breakup time,

ligament length, and the number of satellite droplets are affected the most (Xu et. al. 2014). As

the cell concentration increases, the breakup time increases to 205 s for the 106 cells/mL

bioink, 220 s for the 5 106 cells/mL bioink, and 240 s for the 1 107 cells/mL (Xu et. al.

2014). A higher concentration of cells decreased the length of the ligament formed between the

drop and the nozzle as it fell (Xu et. al. 2014). This reduced the number of satellite droplets, as

the breaking of a longer ligament produced more droplets (Xu et. al. 2014). With cell

concentrations of 5 106 and 1 107 cells/mL, one droplet was formed (Xu et. al. 2014).

Breakup time increases with an increase in cell concentration, as the increased viscosity and the

elastic forces resists the capillary forces that create the break (Xu et. al. 2014). An increase in

cell concentration also decreases the velocity of the droplet due to conservation of energy. When

the drop is ejected, the energy to do so is converted into other forms, such as elastic energy,

reducing energy converted to kinetic energy Xu et. al. 2014). One very common source of non

ideal behaviour this study discovered is that cells accumulate around the nozzle tip, causing

adhesion with subsequent drop (Xu et. al. 2014)s. Thus, when the ligament between the drop and

tip forms, it is forced off center, thereby affecting trajectory and breakup time (Xu et. al. 2014).

This data allows for more ideal and accurate dispensal of the ink during DOD printing,

improving the quality of structure printed.

Creation of a vascular system

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One factor that is limiting the development of more complex printing organs is the

development of complex vascular systems. Of the methods of creation, direct-write technology

allows for freeform fabrication of complex and heterogeneous parts (Christensen et. al. 2015). A

vascular system is critical for the viability of the organ, as it allows nutrients to reach the various

cells, and allows waste product to be removed (Yu et. al. 2013). Two factors that must be

considered is maintaining cell viability and the creation of a complex vascular system. During

the bioprinting process, cells receive different mechanical stimulation, which might affect

intracellular structures and cell membrane integrity (Yu et. al. 2013). As a result, it is critical to

know whether cells can maintain their viability both immediate and for some time after the

printing process (Yu et. al. 2013). The important part of a complex vascular system is the

bifurcations, or branches, of the tubes (Christensen et. al. 2015). Without this, it will be unable to

fulfill its functions throughout the organ (Christensen et. al. 2015).

Yu et. al investigated the effects that nozzle diameter, alginate dispensing pressure, cell

seeding density, and alginate concentration had on the viability of the cells while printing tubular

constructs (2013). The effects of each stimuli were tested by printing a tubular vessel with inner

and outer diameters 13513m and 30922m respectively using additive manufacturing

process (Yu et. al. 2013). The first parameters tested were cell seeding density and alginate

concentration. While there was no significant difference in cell viability of different cell

densities, cell viability decreased with increasing alginate concentration (Yu et. al. 2013). While

2% alginate produced the highest viability at 89%, 4% offered acceptable viability at 68% and

structural integrity (Yu et. al. 2013). Using the 4% alginate concentration, nozzle size and

dispensing pressure were tested (Yu et. al. 2013). Cell viability decreased with an increasing
 3D Bioprinting of Organs 5

pressure and decreasing nozzle size, with cell viability for nozzle sizes of 730m and 550m

92% and 68% respectively, while dispensing pressures of 35kPa, 69 kPa, and 138kPa gave

68%, 51.75%, and 40%, respectively (Yu et. al. 2013).

A vascular system needs both horizontal and vertical bifurcations to create to complex

structure needed (Christensen et. al. 2015). Christensen et. al. investigated a method of freeform

fabrication using a liquid calcium chloride solution as both a support material and a cross-linking

agent (Christensen et. al. 2015). A liquid support allows for simplified removal of the support,

while still providing buoyant and cross-linking forces (Christensen et. al. 2015). A vertical

bifurcation is printed by gradually splitting a straight into two inclined tubes, by printing each

layer slightly overhanging from previous layers (Christensen et. al. 2015). This method

successfully printed a vascular-like structure with both horizontal and vertical bifurcations

(Christensen et. al. 2015). The dimensions of the structures, the mean diameter is 3mm with a

wall thickness of 1 mm (Yu et. al. 2013). In particular, the horizontal portion is designed with a

long tube length of 10 mm, a short tube length of 5 mm, and angles between the branches of 120;

the vertical portion had a straight tube height of 2.5 mm, an angled branch height of 3 mm, and a

90o between the branches (Yu et. al. 2013). One issue that developed is, as a result of the gelled

nature of the structure, horizontally oriented tubes experienced deformation due to gravitational

and impact forces (Yu et. al. 2013). To compensate for this, the printing trajectory was fine tuned

over iterations of printing, until an acceptable structure was made (Yu et. al. 2013).

Applications

While the technology is being improved every day, there are still many current

applications of 3D bioprinting technology. Two general applications ar in regenerative medicine,

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and in in vitro studies (Williams et. al. 2013, Lee et. al. 2015). Its application in regenerative

medicine are in replacing and repairing damaged tissues (Williams et. al, Lee et al.) Its use in in

vitro studies allows for ethical and accurate testing on human organs (Lee et. al. 2015).

Recent studies have shown successful integration of printed tissue organoids created in

vitro (Williams et. al. 2013). As a result Williams et. al. investigated the creation of cellular

soehren created from adipose-derived stromal vascular fraction cells as a proof of concept of the

value of the spheroids (Williams et. al. 2013). These cells were used due to its ability to

differentiate into numerous kinds of cells, including smooth and cardiac muscle cells, adipocytes,

and chondrocyte precursors (Williams et. al. 2013). The spheroids were created by forming a

drop at the end of the printing tip, then lowering the drop into a CaCl2 solution (Williams et. al.

2013). Depending on the size and pressure of the printing head, sheet size varied; with the

18-gauge pen, spheroid size ranged from 1500 to 2500m, while the 23-gauge pen ranged

between 800 and 1700m (Williams et. al. 2013). After printing, the spheroids were placed in

suspension, and integrity, morphology, and viability was measured for 16 days (Williams et. al.

2013). This suspension was successful- not only did the evidence suggest that most, if not all,

forms of suspension would be able to maintain the cells, there was adequate diffusion of

nutrients in and waste out of the spheroids (Williams et. al. 2013).

Another current application is the creation of sections of artificial skin (Lee et. al. 2015).

Skin is the largest organ in the human body, ands its importance cannot be understated, both in

protecting the body and in maintaining homeostasis (Lee et. al. 2015). When the skin is

damaged, skin grafts allow damage to the skin to repair quicker, while performing some of the

functions of the skin (Lee et. al. 2015). Printed skin grafts are more advantageous, due to their
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mimicry of human skin physiology, as well as eliminating ethical concerns from using animal

skin (Lee et. al. 2015). Typically, engineered skin is created by simplifying the tissue into two

compartments: The first is the keratinocytes (KCs), which functions as the epidermis, while the

dermis is simulated with fibroblasts (FBs) (Lee et. al. 2015). Lee et. al. demonstrated the ability

for 3D bioprinting to overcome some of the limitations of traditional engineering (Lee et. al.

2015). Their study demonstrated the ability to create human skin using FBs and KCs as

representative cell types (Lee et. al. 2015). Construction of the skin was done using a layer by

layer fabrication, with a pattern of two layers of collagen in a 6mm square, followed by a 4mm

square of FBs repeated three times followed by two layers of collagen, than two 4mm square

layers of KC (Lee et. al. 2015). One the layers were completed, the skin was incubated for one

hour at 37oC and 5% CO2 to complete the collagen gelation (Lee et. al. 2015). The skin was then

submerged in culture media, with the media changed once every three days (Lee et. al. 2015).

This method was used to print skin on a 12-well transwell membrane support, allowing the

culture to be grown at the air-liquid boundary (Lee et. al. 2015). This allows KC differentiation

into corneocytes (Lee et. al. 2015). The structures were cultured for 4-8 days, allowing for the

grown and development of the KC layers (Lee et. al. 2015). As a method of comparison, skin

constructs were created in the traditional method (Lee et. al. 2015). Lee et. al. found that the

printed skin maintain shape through the culture period, while convention skin constructs started

shrinking on day 2, and were unable to retain their shape (Lee et. al. 2015).

These studies show successful applications of 3D printing. One such application is

regenerative medicine. The spheroids were maintained in culture over long periods of time

(Williams et. al. 2013). As data shows successful integration with existing tissue structure on a
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small scale, this can be used as a method of repairing small areas of damaged tissue (Williams et.

al. 2013). For the creation of skin, 3D printed skin grafts outperformed the conventionally

created one, as it is capable of maintaining its shape over longer periods of time(Lee et. al.

2015). Another application is in vitro studies. The spheroids can be used to mimic tissues of

various type, allowing the effects of chemicals on these tissues to be tested (Williams et. al.

2013). This allows for more accurate data, as there is no change of cross-species differentiation

of data. The 3D printed skin can just as easily be translated to the design of disease models for

autoimmune diseases of the skin such as psoriasis, atopic dermatitis, allergic contact dermatitis,

and vitiligo and models of skin malignancies such as melanoma (Lee et. al. 2015). By using

printed constructs, people can better understand the interactions of various chemicals, deisees,

and drugs within the human body.

Conclusion

In conclusion, the 3D printing of organs is a promising new technology, both presently

and in the future. There are constantly new discoveries on the exact functioning of the processes..

While there are currently viable applications of the technology, such as the creation of superior

skin grafts, or tissue spheroids for studies and regeneration, new developments and

understandings of the process will allow for greater application in the future. Once the

vascularization process is understood and functional, larger and more complex organs can be

created, expanding on current uses. As the use of this technology expands, new questions will be

generated, and new methods discovered. Perhaps, one day soon, those twenty-two lives a day

will be saved, each person leaving the doctors with an organ that was created while they waited.
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