APPLICATIONS OF 3D BIOPRINTING IN DRUG DEVELOPMENT AND

DISCOVERY

Rabita Javeed1, Vidhi Katiyar2, Sandeep Kumar3

1
DST FIST Lab, Sharda University, Plot no 32, 34, Knowledge Park III, Greater Noida, Uttar
Pradesh, India 201310

2
School of Basic Sciences and Research, Sharda University, Plot no 32, 34, Knowledge Park
III, Greater Noida, Uttar Pradesh, India 201310

3
School of Pharmacy, Sharda University, Plot no 32, 34, Knowledge Park III, Greater Noida,
Uttar Pradesh, India 201310

Author for Correspondence:

Name: Dr. Sandeep Kumar Shukla

E. Mail. Id.: ph.sandeep@gmail.com

INTRODUCTION

The process of originating material by depositing material using a printing head, nozzle, and
additional printing technology is known as 3D printing (Kannayiram et al., 2023). According
to the International Standard Organization, 3D printing is also known as ‘additive
manufacturing’. It's a technique for applying material in layers to create structure. But
conventional medicine is reductive, meaning it requires less material to form a
structure (Gungor-Ozkerim et al., 2018).

Researchers have provided detailed descriptions of multiple processes that are included under
the hypernym"3D printing". Since its invention in the late 1980s, 3D printing has seen
significant application in a number of industries, including healthcare. Comparing 3D
printing to traditional manufacturing methods reveal several merits. As it is additive,and it
produces finished goods quickly. Because it makes use of computer-aided designs, complex
objects which can be easily and quickly created. It has been successful in overcoming the
traditional manufacturing paradigm of "one size fits all." The patient's need for customized
medication is one of the main benefits. Furthermore, complex designs and structures can be
designed with the aid of 3D printing. For onsite application, single and multi-drug
combinations can be developed using AM technology. The design of drug dosage forms has
modern, as evidenced by the numerous comprehensive research articles published in the last
few years. According to Moulton and colleagues, 3D printing presents a chance to enhance
drug stability and create hydrophobic drugs with controlled-release and modified-release
formulations. The United states food and drug administration(USFDA) approval of
SPRITAM® (levetiracetam), the first oral disintegrating tablet, is linked to the advancements
in three-dimensional printing in pharmaceutics. The creation of this tablet illustrates how AM
technology can be applied to enhance conventional dosing forms economically for a
prolonged period of time. This will facilitate the development of personalized dosage forms.
Over the past few years, researchers have established teams to develop drug delivery
strategies. One of those businesses that was established specifically to manufacture 3D-
printed medications is Fab Rx Limited. Delivery-related studies have examined implant
administration, trans-dermal administration, and buccal patches (Ali et al., 2023).

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DRUG TESTING AND DELIVERTY THROUGH 3D PRINTING

The use of three-dimensional printing allows for the creation of personalized drug delivery
systems. As a result, 3D printing amplify existing drug screening platforms by precisely
depositing biomaterials containing patient-derived cells and can mimic the natural
environment of a diseased human body (Zhang et al., 2018).

Material extrusion method (ME)

ME-based additive manufacturing allows for the printing of abundant materials and colors on
plastics, food, or living cells. The procedure is widely used, and the costs are affordable. This
process can also be used to create fully functional product components. FDM, or fused
deposition modelling, is the first example of a ME system. Since its inception in the 1990s,
the polymer has served as the principal component of the FDM process. Modelling of fused
deposition structures (Wang et al., 2023).

AM technology used for drug testing

Previously, drug tests were carried out using 2D printed models. They are, however, limited.
The in vitro models that are 2D printed do not perfectly the internal physiological
environment of the body. As a result, they are unable to deliver trustworthy test results. This
problem is being addressed using three-dimensional printed models. Models are classified
into three types:

• Organoids manufactured using 3D printing

• 3D-printed model of an organ on a chip

• Three-dimensional printed model of an organ or tissue

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Drug Delivery and Screening

Bio-printing innovation in RM has made it possible for tissues and organs to develop, as well
as for signalling pathways and other components needed for blood vessel development to
exist.

Figure 1.1, 3D bio-printing in applications in medicine (drug development, screening,

and illness simulation).

Bio-printing of tissues and organs for regenerative medicine

Bio-printing has come a long way, and a variety of tissues are now printed and tested. Many
organ systems cooperate to preserve homeostasis and regular bodily functions. An organ
system is made up of different tissues, organs, and anatomical structures, each of which
serves a particular purpose (Lappi et al., 2023).

Heart tissues

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The heart is a highly segmented organ that keeps blood flowing throughout the body. The
rapid growth of the embryo is facilitated and encouraged due to the development of the
circulatory system and the heart, which happen early in maturation. The heart is made up of
roughly 9 billion cells, including fibroblasts, smooth muscle, epithelium, capillaries, and
other tissue cells. Anomalous extracellular matrix structure or function is the root cause of
heart disease (CVDs).

Innovative in vitro tissue models

There are now in vitro models of organs, tissues, and illnesses, evaluated, optimized, and
applied in tandem with the advancement of 3D bioprinting methods and biomaterials. This
section uses case studies of cancer, the heart, liver, and muscle to analyse the seminal and
exemplary works in these domains (Rastogi & Kandasubramanian, 2019).

Perspectives and scope for the future

3D printing technology will completely change how diseases are treated by validating more
complex, ostentatious drug delivery systems with compatible reactants and more controlled
release profiles. This technology provides unique advantages in terms of product uniformity
customized drug delivery, and combining multiple APIs that improve treatment accuracy for
the patient. Therefore, issues with 3D printing technology will inevitably arise
(Vorndran et al., 2015)
.

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 I. TYPES OF 3D BIOPRINTING

 Major discoveries in bioprinting applications. (4)

Evidently, advanced biotechnology and 3D bio print technologies, which preserve the
potency and viability of various components of cells such as stem cells, have been used
extensively in tissue regeneration and drug development models. This section provides a
detailed elaboration on models of bioprinting for structures such as bone, liver, heart, cornea,
and other tissues associated with the central nervous system. The highlights are in vivo
transplantation, proliferation, maturation, viability, and functionality of cells. Moreover, a
presentation is given on the creation of Biomimetic drug development platforms via
bioprinting. Examples of 3D-bioprinted hepatic, glioblastoma, and cardiac tissue models
further show how 3D bioprinting can boost drug development in personalized medicine. An
outline of the selected key findings is provided.

Printing Organs Differentiated from Stem Cells for Tissue Regeneration.

Bone
The technique of 3D bioprinting for bone regeneration and cartilage repair is becoming
increasingly popular day by day. When it comes to field of bone tissue engineering, a
Scaffold has been designed so that it can be mechanically assisted and will thereby promote
cell processes similar to migration, proliferation or differentiation in lieu of the actual bone.
Due to its biocompatibility and biodegradability, polylactic acid (PLA) is one of the most
widely used materials for scaffolds. They have been a fusion involving the 3D PLA scaffolds
with extracellular vehicles (EVs) as well as human gingival mesenchymal stem cells
(hGMSCs) to test the scaffolds' cytotoxicity and regeneration effects. However, no
cytotoxicity effects were observed with the PLA byproducts breakdown (Zhou et al., 2019).

Rats with damaged cortical calvaria bone tissue showed blood vessels and new bone nodules
in their calvariae subsequent to six weeks of in vivo implantation. In addition, Teixeira et al.

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demonstrated that type I collagen and polydopamine (PDA) surface coatings enhanced the
osteo-inductive properties of three-dimensional printed scaffolds.

Kidney

The kidneys' intricate growth, geographical arrangement, and ancestry requirements have
made bioengineering them difficult. Research has demonstrated that 3D cellular aggregates
produced in vitro exhibit better long-term stability than 2D differentiation. According to
Goulart et al.'s research, bioprinting hepatic tissues with parenchymal cells derived from
human liver induced pluripotent stem cells as three-dimensional spheroids improved the cells'
cellular survival and function in comparison to in vitro single-cell dispersions
(Muth et al., 2014)
.

A significant obstacle in the transplantation of kidney tissue derived from stem cells been the
dearth of nephron architectures. Takasato et al. report that approximately 100 nephrons were
present in a kidney organoid cultured in a transwell, starting with 5 × 105 cells. On the other
hand, 1 ×106 nephrons are thought to be in one person's kidney. An experiment that was
conducted with Lawlor et al. was able to differentiate between bioprinting of kidney
organoids through 3D cellular printing and the generation of renal organs by manual
methods. The cells are manually centrifuged for the formation of an aggregate, and it is then
inserted on top of a transwell filter to produce organoids. Using a NovoGen MMX extrusion-
based 3D cellular bioprinter, cell pastes were automatically deposited to create the organoids
in the bioprinting technique. The proportion of the volume of the deposited cell suspension to
the deposition occurring along the transwell surface, was also adjusted by the researchers to
achieve two distinct bioprinting configurations: a single-point deposition and the other one
comprising a line of cells ~12 mm long. The number of nephrons obtained by fluorescence
imaging in the resulting viable species of organoids was replaced by the MAFB positive
region, while the cell paste was made using the MAFBmTagBFP2 reporter line. They found
that despite having 1.1 × 105 cells at the beginning, the bio printed R40 organoids had a
larger area of glomeruli than the manual organoids, which had 2.3 × 105 cells at the
beginning.

The patch organoid's distribution and functioning were also examined in greater detail. With a
30-deposition ratio comprising roughly about 4 × 105 cells, over a 4.8 × 6 mm total field, cell

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pasted was ejected. Figure 4D shows that the resulting patch had correctly patterned nephrons
and evenly distributed epithelial structures. Another indication relating to functionality of the
nephrons was the TRITC-albumin uptake that was done by the YFP-positive proximal
tubules.

Furthermore, there is a need for research that dive into the bioprinting parameters so as to
increase the nephron numbers that will in turn complement with their human counterpart, the
formation of the nephron in the resulting species, as well as the functionality of the patches in
vivo, provided that there is a greater possibility of modifying the deposition ratios likely to
enhance the formation of newer nephrons and kidney tissues that are of considerable sizes
found in other areas.

Heart

The use of cardiac stem cells to regenerate heart tissue has been attempted numerous times.
Numerous studies have utilized cardiac tissues that have been translated with an implantable
scaffold. But the scaffold's inclusion has raised concerns about immunogenicity,
deterioration, and mechanical properties. As outlined above, there has been a pursuit for an
ideal scaffold to be used. Likewise, a novel method for bioprinting cardiac patches in the
absence of a scaffold has been certainly at hand (Assad et al., 2023).

Cardiomyocytes derived from human induced pluripotent stem cells, fibroblasts, and
endothelial cells were printed into cardiac spheroids in the absence of a scaffold and these
tissues demonstrated both spontaneous beating and the resulting electrophysiological
properties which were similar to the myocytes that were found ventricularly. On top of that,
an in-vivo implantation was conducted using this technique to analyse the potentiality for
regeneration of the tissues. Following myocardial infarction in Lewis naked female rats, bio
printed cardiac patches were inserted, and the scar regions' vascularization and cardiac
functions were evaluated. The implantation group had greater cardiac output, more vessel
counts, and less scar area in comparison to the control group. However, the integration of the
cardiac patch with a native heart was not clearly demonstrated due to some irregularities that
arise between the heart rate of the rats and the contraction rate of a humanist cardiac patch. In
the future, research should be centred around regeneration potentiality studies and integration
particularly on host implants.

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 Neurons and Central Nervous System

Neurons and glial cells are very delicate, making 3D printing to become a bit challenging.
Recent studies demonstrate that human pluripotent stem cells (hiPSCs) which were
particularly immature had been printed then differentiated so as to be assembled into brain
organoids by themselves. This has further shown the co-printing of human neural stem cells
(hNSCs) and biomaterials which upon encapsulation of the stem leads to an original mark of
proliferation and differentiation occurring in functional neurons and neuroglia.

The method used was found to have no control over the differentiation and formation of brain
constructs after printing and several attempts were assembled to print neurons directly. A
different approach based on the extrusion printing of cortical neurons and glial cells using
Matrigel and alginate as the bio-ink was conducted and eventually, neurons continued to exist
for up to 70 days following printing. A network with early and immature activity was also
found by their functional analysis. Aside from developing novel printing technology, other
researchers have also observed early markers of neuronal expression and printed neural
progenitor cells derived from hiPSCs using bio-ink based on fibrin. Additionally,
advancements were made in the implantation of neural stem cells (NSCs) and neural
progenitor cells (NPCs) into biocompatible scaffolds (Tang et al., 2014).

Cancer models in 3D

Apart from bioprinting, several other approaches can be employed to simulate cancer in
three-dimensional environments. Comparing results from various models is challenging due
to the wide range of models that are available.

The choice of cancer model is often influenced by the intended application. Fifty The
literature has described a wide variety of 3D in vitro models, each with unique applications
and advantages. Five One Every approach has benefits and drawbacks. The following
techniques will be carefully looked at: the application of organ-on-a-chip and
spheroids/organoids along with microfluidic devices.

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Organoids/spheroids

The ability of cells that are placed in specialized plates, such as ultra lower microplates to
self-aggregates is essential for 3D modelling tumours without the need for scaffolds. These
aggregates, also called spheroids or organoids, exhibit traits that are similar to those of in
vivo tumours, including cellular heterogeneity, secretions from cells, and kinetics of volume
growth. Spheroids extracted from specific cancer cells that are able preserve their genomic
structure facilitate drug development and patient-specific testing. The hanging drop method
encapsulates cells in media drops made with a specialized plate, causing the cells to self-
aggregate that is caused by the absence of adhesion surface (Choonara et al., 2016).

Microfluidic organ-on-a-chips

These devices recognize and structures the proximity existing between normal parenchymal
cells and ECM in the local tissues which thereby affects the growth of various types of
cancer. These chips guide a promising direction towards cancer detection, drug screening and
cancer modelling in the recent years. Using three dimensional in-vitro systems in comparison
with two dimensional in-vitro systems offers a cleaner slate of viewpoints in that similar
direction.in other words, conventional 3D devices only partially support cell to cell
interactions and structures that are located spatially. Furthermore, the efficiency of a system
as a powerful screening platform is however limited by the larger volumes of samples
required. These systems when developed to fit in a specific dimension may offer better
quality of biological models while also reducing the necessary costs thus making them more
and more popular (Aimar et al., 2019).

Conventional 3D systems are designed to become more functional and efficient by using
microscale 3D in vitro models allowing the development and modification of microfluidic
cancer models to turn into a beneficial approach towards cancer research. Microfluidics uses
tiny devices with channels that range in size from a few tens to hundreds of micrometres to
adjust smaller volumes of fluids. The process of creating microfluidic chips usually involves
creating tiny wells or thin grooves on the surface, which is subsequently surrounded by
another layer bonded to the surface to form a chamber comprising of microchannels which

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are required to be leak-proof. Nevertheless, organ-on-a-chip models are unable to accurately
replicate the tumor due to their small size.

3D bioprinting

In tumor biology, it requires a three-dimensional (3D) microenvironment which allows the

replication occurring between tumor cells and their surroundings. 3D bioprinting moves in
promising direction towards creation and development of cancer models due to the privilege
that allows such replications and diverse cultures which are placed under controlled chemical
and mechanical conditions. Using a bottom-up assembly method, it deposits biomaterials and
cells in predetermined patterns to create 3D biological structures layer by layer. Although a
number of bioprinting techniques have already been applied to these applications, extrusion,
droplet, and laser-based technologies stand out the most (Li et al., 2021). Below is a summary
of such methods; figure 1.2

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The widely used method of extrusion-based bioprinting shapes bioinks into desired patterns
by means of air pressure, mechanical pistons, or screws. This process is reasonably easy and
economical, and it can be used with a large range of materials.

Lesser survival rates and diminished functionality of the printed tissue may arise from the
pressure exerted on the cells during the process. This method is flexible in the field of cancer
research because it can generate models that resemble the architecture of in vivo tissues and
depict various cancer stages, facilitating a thorough comprehension of the illness and possible
therapeutic approaches.

Bioprinting for use in plastic surgery

The intricacy and precision of 3D bioprinting products must similarly be linked to the body
tissues and organ structures, along with multi-functionality that occurs in the human body.
There are significant differences in the composition and capabilities of various human body
tissues and organs. Consequently, several technologies that are associated with 3D bioprinting
continue to surface so as to meet the need for printing intricate structures at a higher precision
that may replicate various body tissues and organs.

The primary 3D biotechnologies available today are:

1. Laser-based bioprinting technology

2. Inkjet droplet-based bioprinting technology

3. Using extrusion technology for bioprinting

4. New developments in 3D bioprinting technology

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 Bioprinting technology based on inkjet droplet

Figure 1.3, A schematic of a bioprinting technique using an inkjet.

(i) Bioink is splitted into constant droplets that are influenced by the surrounding electric
field in addition to their own charge.

(ii) You can create patterns by ejecting droplets in any direction using Inkjet printing with
drip-on-demand, which employs three distinct drive technologies: electrostatic, piezoelectric,
and thermal. Figure B schematically illustrates the Stereo Lithography Appearance method.

D. A laser-assisted bioprinting method's schematic diagram. D. Schematic illustration of an

extrusion-based bioprinting method (Tan et al., 2016)

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 Emerging 3D bioprinting technologies

Figure 1.4, A schematic of a new 3D biological printing strategy.

A schematic representation of the conventional embedded bioprinting approach is

provided.
B Diagram of the microfluidic bioprinting strategy.

(i) a coaxial flow focused extruder,

(ii) a microfluidic print head with a pneumatic valve, and
(iii) a coaxial flow focused extruder. B A schematic representation of the traditional
volumetric bioprinting method is provided.

In plastic surgery, Human tissues and organs can have flaws and deformities fixed and
rebuilt., and the normal form of human is reshaped to enhance and beautify form and restore
function. Surgical techniques such as tissue and organ transplantation are used in this process.
Repair and reconstruction of various organ tissues are the focus of plastic surgery. The
challenges faced by plastic surgeons are constantly increasing due to donor area destruction,

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pain, deformity, and infection related to the use of (autologous and allogeneic) grafts in
clinical surgery.

In addition to printing various operational cells, extracellular matrix (ECMcell growth factors
and support materials made of biodegradable polymers, 3D bioprinting enables the creation
of individualized and tailored implants according to patient requirements, including in situ
printing that takes place right on the patient's injured area to aid in wound healing. The
development of these technologies has allowed for true precision medicine, prevented
surgical aftereffects, and significantly decreased the difficulty of surgery (Yang et al., 2017).

In addition to printing different functional cells, extracellular matrix (ECM), cell growth
factors, and biodegradable polymer support materials, 3D bioprinting enables the creation of
individualized and tailored implants according to patient requirements, such as in situ
printing that is done straight on the patient's injured area to aid in wound healing. With the
advent of these technologies, surgery is now much less difficult, surgical aftereffects are
avoided, and true precision medicine is made possible. Important fields of plastic surgery that
benefit from this technology include rhinoplasty, breast implantation, ear reconstruction, skin
regeneration and healing, and maxillofacial bone repair.

Lab-On-The-Chip

The potential of bioprinting in conjunction with lab-on-a-chip applications is one of its most
intriguing features. It produces living micro-organs that resemble actual human organs
remarkably well, on size of microchip. To fully realize the prospective of bioprinting in the
area of lab-on-a-chips, one must have a thorough understanding of these technologies. In
essence, they are microchips that contain chambers, channels, microfluidic elements, and
cells which can replicate biological events and stimuli through a span of mechanical events
that are artificially induced. It is possible to optimize a wide range of stimulus and chip
characteristics, including stiffness, oxygen gradients, flows of fluids and additional chemical
stimulants.

Drug screening makes extensive use of on-the-chip technology, especially when it comes to
organs such as the kidney and liver. Organs-on-a-chip, which are utilized to investigate lung
functionality, also aim to replicate the stretching motion that results from breathing.
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Moreover, the precision of the system depends on bioprinted 3D component of this
application, which prints an organ or tissue structure with native cells, a similar physical
structure, and a precise extracellular matrix.

Skin replication

Replicating human skin is a significant use of 3D bioprinting since it is an essential tissue that
shields the body from the outside world. Skin replacement with grafts is necessary to keep
safe the body and stop water loss when this essential tissue is severely damaged by burns or
wounds. Skin replacement is currently carried out using skin grafts. Grafting will eventually
be superseded by the tissue engineering (TE) field. Treatment with TE may also be possible,
as it can be used to study the characteristics of the skin, including its permeability and
response to different substances. Bioprinting 3D is a perfect technique for producing more
precisely printed skin because of multilayer and multicellular human epidermis
structure (Mirzoeva et al., 2018).

Bones

Artificial bone manufacturing is now heavily reliant on 3D bioprinting. The main application
of 3D printing technology for bone repair prior to bioprinting was the creation of ceramic
scaffolds and other similar materials. For this purpose, synthetic polymers like PCL and PLA
were also widely employed; however, the processing temperature of these polymers was a
constraint, which made encapsulation of cells challenging. Because of these material-based
limitations, the absence of local bone cells usually limited biocompatibility.

To address these problems, biopolymer hydrogels—like chitosan and alginate—have been

researched for application in 3D-printed bones because of their well-established
biocompatibility, high water content, and structure that resembles extracellular matrix and
permits cellular integration.

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Ophthalmology

3D bioprinting uses in ophthalmology is another area of interest. This application is critical

for the immunity of eye as well as in the ease in which doctors may examine ocular
conditions using simple imaging techniques. The cornea is the focal point for any 3D
bioprinting applications in the human eye, as it is the one of the most commonly organ
transplants that are performed in the United States and found rather expensive in other parts
of the world. As a matter of fact, over 40,000 cornea implant surgeries are performed each
year in the United States alone. Although there are various allogeneic grafts available for this
transplant, the post procedure condition is a setback due to the issue of immune rejection.

3D bioprinting has been explored for the creation of lenses and retinas in addition to the
cornea. Creating a refractive index using 3D bio-printed lenses is the most challenging task.
Moreover, PMMA filaments lack the flexibility necessary to form intraocular lenses with
such characteristics. Conversely, silicone presents a viable substitute material due to its
flexibility, transparency, and suitability for 3D printing. The utility of live-cell printing to
induce changes in the refractory structures warrants further investigation as it may prove
beneficial in a range of future applications. In contrast, a great deal of research has already
been done on 3D bioprinted human retinas. A technique for generating a functional retina
from retinal ganglion cells and glial cells was presented using a rat. In order to preserve the
cells, the researchers employed a piezoelectric inkjet printer, which is a potentially useful
method for designing retinas which were better biocompatible for transplantation
(Su et al., 2021)
.

Bladder

3D bioprinting has also potential applications in tissue regeneration and future transplants for
this organ. Bladder issues are often resulting from surgery performed on other parts of the
body and can have a deleterious effect on the extracellular matrix. However, bladder issues
may also be caused by inflammation, trauma, cancer, and body infection. However, the most
common reason for bladder removal (a cystectomy) is cancer. A male cystectomy usually
involves the elimination of the prostate, lymph nodes, ureters, and seminal vesicles. In
women, it means eliminating the lymph nodes, ureters, uterus and the vagina. The
intrusiveness of this therapy is however increased when one considers the population afflicted
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by this illness. The autonomic and sensory nervous systems regulate both the muscular wall
and the urothelium, which make up the functional bladder. These elements are necessary for
the proper operation of the bladder.

Urine is generally stored in the urothelium at high capacity and low pressure. The bladder's
primary purpose of preventing potentially harmful metabolites from entering the bloodstream
and hypertonic urination can be fulfilled by a combination of these functions. The urothelium
is primarily composed of basal cells, but it also contains some intermediate and superficial
cells from the luminal layers. Furthermore, the bladder system cannot operate without the
smooth muscle system, innervation, and an active vascular system. Considering each of these
factors is necessary to complete the 3D bioprinting process and create an artificial bladder
that is both effective and biocompatible (Kumar & Ranjan Sinha, 2015).

Kidney

Recent developments in the differentiation of Pluripotent stem cells in humans have made the
three D bioprinting of kidney a feasible option for drug screening and modelling. It is
commonly known that the kidneys regulate blood pressure, differentiating substrate levels in
the bloodstream, and eliminating toxins from the circulatory system. Owing to these vital
roles, the kidney should be taken into account when evaluating medications that the body
might deem hazardous. Human pluripotent stem cells were first used by Czernieck et al to
create kidney tissue in a 96-well format. After distributing the cells on a Tecan liquid
platform, nephron tissue was produced. Using this information, Higgins et al. produced
kidneys from roughly 4000 differentiating stem cells in a second study on the 3D bioprinting
of kidneys using a NovoGen MMX bioprinter. The creation of an artificial kidney and its
replication, along with its morphology and cellular equivalency with natural kidneys,
demonstrated the experiment's success and allowed for its potential use in drug screening
tests following printing. In 3D bioprinted kidneys, however, the most important factor is the
morphology's accuracy and precision.

By the time the printing was finished, the epithelium, renal, stroma, and endothelium,
glomeruli are all important kidney organelles—were all visible. The study also demonstrated
the possibility of producing multiple organoids from fewer starting cells, which is an
important lesson. To create certain organoids, as few as 4000 human pluripotent stem cells

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were required. Additionally, to show that fluorescent reporters lines are viable for use in
medical imaging, they were used as indicators on a variety of kidney models that were
developed. In conclusion, this research opened up new directions for this kind of study and
showed that creating artificial kidneys for use in human patients is a feasible future
endeavour.

For drug screening, synthetic 3D bioprinter models of the liver could be helpful as it is
another metabolic organ. Approximately 75% of the liver's cells are hepatic parenchymal
cells, making them the most important type of liver cells. Such 2D models and cultures of
these hepatocytes have been used in previous in vitro drug screening studies, but the
outcomes have not been particularly promising. The development of 3D modelling and
bioprinting was spurred by these setbacks, which forced researchers to search for alternative
in vitro drug screening methods. Transwell culture-based, micropatterned, spheroid, and
scaffold-free model structures are the most widely used types for 3D bioprinting.

Because the micropatterned models show promising results in liver toxicity and metabolism,
they have been studied and repeated the most among these options.

Without the use of a scaffold, Kizawa et al. attempted to print a liver in three dimensions
using HMCS1SA hepatocytes and mouse fibroblasts. The lab created nine spheroids with a
complimentary Regenia printer, which fused over a week to form the liver tissue. Following
the fusion, the liver tissues underwent 20 days of post-plating experiments and two weeks of
culture. The hepatocytes' gene expression on day 22 matched that of day 0 according to
microarray analysis.

Because these genes are involved in liver function, including the metabolism of drugs,
glucose, lipids, and urea as well as the synthesis of essential serum proteins, this was crucial
to the experiment. Furthermore, to evaluate the efficacy of detoxification, CYP3A4 mRNA
expression was measured using CYP3A4 enzyme activity, which also demonstrated function.

It is possible to conclude that liver models could be useful in vitro and that the creation of
artificial liver transplants might not be too far off (Kumar & Ranjan Sinha, 2015).

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Pancreas

Nephropathy, tingling brought on by peripheral nerve neuropathy, loss of peripheral

circulation in the extremities, and visual impairment are among the symptoms of this illness.
Including associated illnesses, diabetes is the eighth leading cause of death globally, directly
responsible for 1.5 million deaths.

Type 1 diabetes mellitus accounts for about 10% of all cases of the disease and is caused by
an autoimmune dysfunction that reduces the ability of pancreatic β cells to generate insulin
which is Currently the only treatment available for diabetes mellitus type 1 and other
variations of the disease is administrating on daily bases. Therefore, its crucial to analyse
simulated pancreases that could be used as future implants and in vitro studies.

Our comprehension of organ's operations and the drug’s effectiveness can both benefit from
this type of analysis (Ahmad et al., 2020).

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Future applications and limitations: 4.D bioprinting

Constructing vasculature, processing time, and material availability and selection are the
main conditions of 3D bioprinting. Since the choice of material and bioprinting technique
significantly impacts a printed structure's functionality, there is no absolute material for any
particular use. Although, bioinks can be customized by incorporating different components.
For instance, a bone tissue application could use hydrogel containing tricalcium phosphate or
hydroxyapatite When it comes to 3D bioprinting, processing times are lengthy and
manufacturing efficiency is low. The longer print and processing times can have a negative
influence on cell viability by changing signaling pathways and cell geometry.

Solutions to these issues lie in creating buffers to aid in cell protection during the bioprinting
procedure and improving existing bioprinting methods to reduce processing times.
Vascularity, which is necessary for a bioprinted tissue to successfully integrate with native
tissue, is the final major constraint. Techniques for vascular bioprinting are often labour-
intensive to process and have poor resolution. Cell survival is frequently low when printing a
more complex structure with vasculature because it takes a long time. Adding angiogenic
factors to the bi-oink to encourage host vasculature growth or using a bioreactor to extend
cell life while vasculature develops are the two workarounds for this.

In terms of prospective applications, 4D bioprinting may eventually be used in medicine in

place of 3D bioprinting. 4D printing considers changes to the surrounding environment that
affect the cells, as opposed to t static features of 3D printed tissues and the body organs.

Instead of taking into account the fourth dimension, 4D printing capitalizes on the time it
takes for extracellular matrices to form in printed cells. Two 4D bioprinting types are printing
new materials and based on maturation. Ingenious or innovative media employ the bioink's
sensitive components to trigger changes in response to stimuli from the surroundings.

These transformations include, for example, self-folding and assembly-disassembly.

Applying both internal and exterior stimuli, such as matrix deposition, cellular coating, and
self-organization, to the first 3D printed tissue is known as maturation-based printing. These
concepts have the potential to significantly impact how effectively the human body can
regenerate tissue in the long run (Borremans et al., n.d.).

Though bioprinting 4D materials are less common because They must possess both bio-
compatibility and stability, 3D bioprinting materials come in a wide variety. The preservation
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of these two characteristics through ideal water and temperature conditions as well as
magnetic fields is essential for complex organs such as the liver and heart. The most
important factor taken into account when building artificial structures is thermal
responsiveness, which is used to contract, fold and swell the particular organ. The thermo
responsive materials poly(~-caprolactone) and poly(N-isopropy-lacrylamide) are two
examples. Water-sorption is a parameter that is equally important for creating an organ that is
biocompatible with the human body because it can be used for both shrinking and swelling.

In addition to outside stimuli, Matrix deposition, cellular coating, and cell self-organization
are additional components that can facilitate successful 4D bioprinting. Vascular graft
endothelial cells are an example of an efficient cellular coating because they have shown
promise in the prevention of thrombosis. HUVECs were injected into implementation, and
then the resultant vasculature was observed after a two-day incubation period. Cell self-
organization has demonstrated potential for generating more lifelike models of human
smooth muscles by utilizing cellular spheroids to form ring-like grafts. It is attained by
efficiently setting the toroid or spheroids in motion.

Administering drugs and tissue engineering are two common applications of 4D bioprinting.
Tissue engineering is unable to effectively address the problem of vascularization, or the lack
of it. The preservation of cellular functionality requires artificial structures with blood vessels
that are bio-accurate, particularly in light of the growing demand for organ transplants.
However, layer-by-layer bioprinting can solve this problem because it creates the first
cylinder-shaped structures, which can be developed by cells and maturation factors like
MSCs, fibro-blasts, and endothelial cells. Drugs and cell release can be precisely controlled
by 4D bioprinting since it can be used to precisely control the placement of different elements
that can be shaped to alter their shape.

One method to accomplish this is to use water-based droplets to create bi-layers.

Nevertheless, they have the ability to alter their form and release the object they are carrying
if placed in a novel environment with varying temperatures and pH levels. Multisomes and
swell-able polymeric hydro gels are two more drug delivery use of 4D bioprinting.

The potential for bioprinting 4D to advance 3D bioprinting means that the medical
community should be hopeful about a future in which there will be a plentiful supply of
organs that are bio-compatible and ready for transplant (Ascione et al., 2023).

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PAGE \* MERGEFORMAT 2
Conclusion

We thoroughly examined the field of live-cell 3D printing and its uses in 3D bioprinting in
this paper. Initially, we talked about the several ways that drug delivery and testing are crucial
to the drug development and discovery process. This paper also discusses the various forms
of 3D bioprinting and how they compare to present and upcoming technique models in 33
bioprinting applications.

In contrast to alternative 3D cell culture methods, 3D bioprinting technology can provide a

degree of biomimicry, architectural control, and customization. All things considered, 3D
bioprinting may result in the creation of a novel biophysical technique for tumor spr
prediction as well as a ground-breaking but highly successful morpho dynamical hallmark of
tumor severity.

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