Lec 15

INDIAN INSTITUTE OF TECHNOLOGY ROORKEE
NPTEL
NPTEL ONLINE CERTIFICATION COURSE
Biomedical Nanotechnology
Lec - 15
Nanotechnology in Organ Printing
Dr. P. Gopinath
Department of Biotechnology
Indian Institute of Technology Roorkee
Hello everyone I welcome you all to the 15th lecture of this course. The 15th lecture is on nano
technology in organ printing. So in the previous lectures, we have leant how to make artificial
tissues and artificial cells. In today’s lecture we are going to learn how to make artificial organs
using organ printing technology.
(Refer Slide Time: 00:39)
In this lecture we are going to learn what is organ printing, types of 3D printing and what are the
various 3D bio printing approaches available. We are also going to learn about the role of nano
technology in organ printing.
Why do we need organ printing? According to the American organ donor website, at least
120000 people are in the waiting list for organ transplantation and each day at least 22 people
lose their life due to lack of suitable organ transplantation. So you can think about the whole
world as how many people are losing their life without suitable organ transplant.
Let us see the statistics in India. In India almost 1.5 lakh people need a kidney transplantation.
But only 3000 of them receive it and 90% of people in waiting list die without getting an organ.
India’s annual liver transplant requirement is 25000 but we manage only about 800 and 70% of
liver transplants ate taken care of by a live donor and remaining 30% are dependent on cadaver
donation.
Organ printing is done by integrating the biology and 3D printing technology. It is a process
where an artificial organ can be created using a 3D printer or a bio printer. The organ printing is
a rapid prototyping computer aided 3D printing technology. It is based on layer by layer
deposition of cells on a 3D gel with a sequential maturation of the printed construct into perfused
and vascularized living tissue or organ. So in this organ printing, we print artificial organs using
the 3D printing technology and use biological cells as inks. We will be printing the organ in a 3D
environment and make the complete artificial organ.
The organ printer incorporates two technologies- tissue engineering and 3D printing. It is similar
to a normal printer but instead of paper we will be using the Petri dishes and instead of ink we
will be using cells and chemicals called as cross linkers. Here we are going to take the cell from
the patient’s own body and so there won’t be any immune rejection of the organ.
There are 3 types of 3D printers. The first one is the selective laser sintering. The second is the
thermal inkjet printer and the third is fused deposition modeling. The selection of 3D printers is
based on the material which you want to use and also how the layers in the finished product are
bonded.
Let us see first discuss about selective laser sintering. The SLS printer uses powdered material as
a substrate for printing new objects. The laser draws the shape of the object in the powder and
fuses it together.
Next is thermal inkjet printer. The inkjet printing is a noncontact technique, which uses the
thermal electromagnetic or piezoelectric technology to deposit tiny droplets of ink. The ink can
be an actual link or it can be any another material onto a substrate according to the digital
instructions based on which the printer will make the organ or scaffold.
The third method is the fused deposition model (FDM). Here the printer uses a print head similar
to an inkjet printer but instead of ink the beads of heated plastic are released from the print head
as it moves and this builds the objects in thin layers.
Let us discuss about the steps involved in the process of bio printing 3D tissues. The first step is
imaging. If you want to make a particular organ like heart or liver, you have to do the imaging
using X ray, CT scan and MRI so that you will get the complete idea about the organ. Then
based on the image, we have to select the design approach. The design approach can be
biomimicry or self-assembly. Once you choose the design approach, we have to select the
suitable material.
Depending on the organ that you want to print, you have to select the synthetic polymers or
natural polymers which will mimic the extra cellular matrix. The fourth step is selection of cells.
The cells can be differentiated cells or it can be stem cells. You can add the cells onto the
scaffold and make the particular type of organ. Once you select the material as well as the cell
type, then you have to bio print that particular organ using the inkjet printer or micro extrusion or
laser assisted printers.
After the particular organ is printed, they are placed in the bio reactor and allowed to mature.
After maturation in the presence of chemical and mechanical signals, the organ will be tested for
its function and finally it can be transferred to the patient.
Now let us see the steps in detail. The first step is creating a blueprint of the organ with its
vascular architecture. The second step is generating a bio printing process plan and 3rd one is
isolating the stem cells or any other suitable cells. The 4th step is differentiating the stem cells
into organ specific cells. The 5th step involves preparing the bio ink reservoirs with organ
specific cells, blood vessels and support medium and loading them into the printer. Then the
complete organ is printed and placed in a bio rector prior to the transplantation. These are the
various steps involved in the bio printing of 3D tissues or organs.
So how the organ printing works? The procedure of organ printing can be subdivided into 3
sequential steps. The first step is pre-processing, next one is processing and third one is post
processing. So let us see these step in detail.
In the pre-processing step, the first is bio imaging the particular organ. Based on the CAD, you
will be making the blue print of the particular organ. In the second step, that is the processing
step, you will be using bio inks. Here the bio ink is your cells and the bio paper is the scaffold.
So based on this bio ink and bio paper, you will print the particular organ using the bio printer.
Once the organ is printed, you will be adding the maturogens, which includes the chemical or
mechanical signals to make this artificially printed organ to work exactly like the real organ.
Then the printed organ will be monitored in the bio rector and assessed for its function. This step
is called as post processing.
Step 1 is pre-processing. The pre-processing step primarily deals with the development of
computer aided design that is CAD or blue print of a specific organ. The design can be derived
from the digitized image reconstruction of a natural organ or tissue and the imaging data can be
obtained using MRI or CT scan of the particular organ.
Then using this computer aided design you will make the blue print of the particular organ. This
step is a pre-processing step.
And step 2 is processing. The processing usually refers to the actual computer aided printing or
layer by layer placement of cells into a 3D environment using CAD or blue prints. So here you
will be printing that particular organ using the bio printers. Instead of paper you will be using the
petri dishes, which is filled with the water. So when the printer prints, the cross linkers transform
the water into jell–O like substance and this allows the cells to be put in. Once one dish is filled,
a new one is replaced on top of it.
This method is repeated until the organ that you want is created. Once the organ is constructed,
the petri dishes are removed and all that is left is the organ. Here the supporting biocompatible
gel is the bio paper and the cell is the bio ink. The cells will be printed on the particular bio paper
layer by layer. When one layer is finished, you will keep another layer of bio paper and again
print the cells. So it is layer by layer assembly of cells. Once all the cells are fused together, then
you can remove this bio paper.
Here the bio ink spheroids are printed into layers of bio paper gel. The additional layers can be
printed to build the object. Once the cells are fused, you can remove the bio gel or bio paper and
you will get the final living tissue.
Let us see this in detail. The organs could be built layer by layer by printing clumps of cells onto
a gel that turns solid when warmed. Once the cells have fused, the gel can be removed simply by
cooling it. Hence the cells are printed on a thermo reversible gel. It will remain in the gel form
when this is warm and after printing it can be removed just by cooling it.
This is your bio paper. On top of the bio paper, you are going to print the cells. Once it is printed,
you can put another paper on the top and you can print another layer of cells. After printing all
the cells, they will be fused together. Once it is fused, we can remove the bio paper by simply
cooling it. At warm temperature, it will form a jelly like substance. It is a thermo responsive gel
and so when you cool it, you can easily remove the bio paper and finally get the desired organ.
The bio printers have three major components. The first one is the hardware used. The next one
is the type of bio ink and the third one is the material on which it is printed. The bio ink is a
material made from living cells, which behaves much like a liquid and allows people to print in
order to create a desired shape. To make the bio ink, researchers create a slurry of cells, which
can be loaded into a cartridge and inserted into a specially designed bio printer. It will be printed
on a gel like substance that is your bio-paper.
The bio ink, as I told you earlier, these are cells that are prepared by mixing cells with bio
compatible materials. For example, suitable hydro gels are selected based on the organ which
you want to print. Some of the bio ink materials that we can use are collagen, alginate or fibrin.
These materials have very good bio comparability and they can homogenously incorporate cells
and growth factors. They are processed under mild conditions. Moreover they allow easy
chemical modifications and sol-gel transitions.
These are various examples of bio paper. We can make a thermo sensitive bio paper based on
agarose, collagen and gelatin. We can also make pH sensitive bio paper that is on HPMC and
also ionic cross linking alginate and photo polymerizable, PEG based or PVA based bio papers.
Out of these, fibrin hydro gel and alginate hydro gel are the very good materials for bio
fabrication.
The fibrin hydro gel has very good cell attachment, cell proliferation, and also cell differentiation
properties. The alginate hydro gel has very good mechanical properties and also it supports the
3D structure to arrange the cells, proteins and growth factors.

Let us see the types of bio printers. There are three types- inkjet printers, extrusion printers and
laser assisted bio printers. Let us see them in detail.
The first one is inkjet printers. These are mainly used in bio printing for fast and large scale
products. They are called as drop and demand inkjet printer because this printer print materials in
exact amounts and so it will minimize the cost as well as the waste. The inkjet bio printer is
divided into two types-thermal and piezoelectric. In thermal inkjet printers, electrical heating of
the print head produces air pressure pulses which force droplets from the nozzle. The acoustic
printers uses pulses formed by piezoelectric or ultrasound pressure, based on which cells are
deposited on the particular bio paper.
The next type of bio printers is the extrusion printers. The extrusion printers print cells layer by
layer. It is also like the 3D printing and in addition to just cells, here the extrusion printers may
also use hydro gels infused with cells. The micro extrusion printer uses pneumatic or mechanical
like piston or screw dispensing system to extrude the continuous beads of materials or cells.
The third one is laser assisted bio printer. Here the printer utilizes lasers and the laser provides
high resolution printing. But this printers are little bit costly and it uses lasers to focus on the
absorbing substrate to generate pressure so that propel this cell containing material onto the
collector substrate.
Let us see the comparison of the types of bio printers. Depending on the organ which you want
to print and the material to be used, the suitable bio printer has to be selected. Each bio printer
has its own advantages and disadvantages. For example, in the case of cell viability, the laser
assisted bio printer has more cell viability and in case of printing speed, the inkjet printer is very
fast when compared to the other two types. In case of printer cost, the inkjet printer costs less and
the micro extrusion is medium in cost and the laser assisted printer is very high in cost. So you
have to select the bio printer according to the type of organ that you want to print.
Now let us see the ideal material properties for bio printing. The selection of appropriate
materials for using bio printing and their performance in a particular application depends on
several features. The first one is printability that is the properties that facilitate handling and
deposition by bio printer and it may include the viscosity gelation methods and rheological
properties.
The next one is bio compatibility. The printed organ should be compatible to the biological
system. The material used should not induce any undesirable local or systemic responses and it
should also contribute actively and controllably to the biological and functional components of
the construct.
The next one is the degradation kinetics and the byproducts. The degradation rate should be
matched to the ability of the cells to produce their own ECM. So before the scaffold degrades,
the particular cell should make its own extra cellular matrix and the degradation by products
should be non-toxic. That means it should be bio degradable and once the scaffold is degrading,
it should not induce any toxic or immune responses in the body.
The fourth is the structural and mechanical properties. The material here should be selected
based on the required mechanical properties of the construct. They can be rigid thermo plastic
polymer, fibers for strength or soft hydro gels for cell compatibility etc. So depending on the
organ that you are going to print, you have to select the particular material.
The fifth one is important that is the material bio mimicry. The bio mimicry requires the
duplication of the shape, frame work, and the micro environment of the particular organ and
tissues. The application of bio mimicry and bio printing involves creating both identical cellular
as well as extra cellular parts of the organ. So in the bio mimicry approach, we have to create the
same environment.
The third step is the post processing step. So the post processing is concerned with the perfusion
of printed organs and their biomechanical conditioning to both direct and accelerate the organ
maturation. To maintain the organ properties, both mechanical and chemical simulations are
needed. These stimulations send signals to the cells to control the remodeling and growth of
tissues. In recent development, bioreactor technologies have allowed the rapid maturation of
tissues, vascularization of tissues and the ability to survive the transplants.
So here the bio reactors work either in providing nutrient transport or it can create the micro
gravity environments. Each type of bio reactors is ideal for a specific type of tissue for example
compression bio reactors are ideal for cartilage tissue.
So this is your example of post processing, okay so here this artificial printed heart is growing in
a bio reactor.
The components of post processing are maturogens and bio monitoring. In the bio reactor, we are
going to keep the artificially printed organs. The maturogens means chemical signals or
mechanical signals, which will provide the real environment to the artificial organs. In bio
monitoring, we will be monitoring the metabolism of the artificially printed organs, whether it is
performing like the original organ.
Recently scientists have created a new electronic membrane, which could replace the pace
makers. They made a membrane using a spider like web or network of sensors and electrodes,
which will continuously monitor the heart’s electrical activity. In future, it could deliver
electrical shocks to maintain a healthy heart rate. Here the researchers used a computer modeling
technology and printed this prototype membrane. Then they fit that into a rabbit’s heart and
checked whether it is working properly. In the rabbit heart outside the body in a nutrient and
oxygen rich solution, it was working perfectly like a real heart. So in future it could be useful for
application in humans also.
Let us see some of the other human scale bio printed tissues.
The first one is two dimensional tissue. Like the skin and cartilage, this was already printed using
the inkjet bio printing systems by Wake forest institute for regenerative medicine. Similarly they
also printed the heart valve and trachea using the micro extrusion bio printed technique. They
have also printed solid organs like kidney using laser bio printing as well as micro extrusion bio
printing technology.
Here the time frame for the development of 3D bio printed tissues depends on the organ that you
want to print. In case of the two dimensional tissues, it requires very less time and in the case of
solid organs, it needs more time.
So as the complexity of the tissues increases, new approaches will be needed to overcome the
challenges of creating them by bio printing. The 2D organs have already been fabricated and
tested, and these will be likely to be one of the first prototypes which could be transplanted in the
patients. And next one is hollow tubes including blood vessels. Scientists have already developed
the hollow tubes and it is now in the clinical trials. The third is the solid organs. These are the
most complex, and so there are still many challenges to overcome, especially in achieving
vascularization and innervations.
Let us see what the challengers are. Research groups have printed the artificial kidney in seven
hours but these are not functional in human. So why doesn’t it work? Because it is difficult to
create blood vessels between tissue layers and organs have many specialized functions difficult
to replicate. So this team came up with some new solutions to overcome these problems. They
created a sugar template that can help shape the development of a vascular network for artificial
organs. After the network is printed, cells are inserted and the network then grows. The sugar
template is dissolved after the completion of development.
Let us see the role of nanotechnology in organ printing.
We can bio fabricate the Janus like tissue spheroids using the microfluidics. A Janus particle
means these are special types of nanoparticles whose surface have two or more distinct physical
properties. So in this case a Janus like spheroids are the clumps of cells having the magnetic
properties.
Using the magnetic properties, we can assemble these particles using an external magnetic field.
Once assembled in a particular shape, these cells will fuse together and form the particular organ
or the particular tissue. We can see here at zero hour, all these tissue spheroids are assembled
using the magnetic field. At fourteenth hour it forms a branched structure as a result of fusion of
closely placed tissue spheroids.
Let us see how we can make technologies for magnetic levitation of tissue spheroids. The first
approach is that we can have the cells labeled with magnetic particles. The second approach is
we can have the tissue spheroid into hydrogel containing magnetic nanoparticles. The third
approach is encaging the tissue spheroid in magnetic micro scaffolds.
We can make the lock and hook like structures and we can make kinds of lockyballs. The
lockyballs are inter lockable micro scaffolds for encaging the living tissue spheroids. By using
this approach, we can make different kind of shapes and in this we can load the tissue spheroids.
We can see here this is the section of lockyballs which is having magnetic nanoparticles inside it.
The red colored are the magnetic nanoparticles. This is the lockyball with magnetic surfaces and
the third one is lockyball with the functionalized magnetic nanoparticles on the surface.
Using the robotic tissue spheroids biofabricator, we can make micro scaffolds that is the
lockyballs in a molded agarose hydrogel. We can seed cells in that and we can make the tissue
spheroid formation in the lockyballs.
Let us see the design elements of clinical robotic bio printers. Based on the lockyballs, we can
make clinical robotic bio printer. Here this one is the tissue spheroid harvester. It is based on
magnetic levitation of tissue spheroids. And here we can use that and make the clinical robotic
bio printer. The tissue spheroids are harvested from the multi wells and it can be translocated and
dispersed into the living tissues using magnetic levitation.
This is a computer aided design of nozzle of clinical robotic bio printer. It contains several
channels. Among them two channels are for fibrinogen and thrombin, and one channel for tissue
spheroids. An additional channel is for pressurized air to enable fibrin hydrogel spraying.
Similarly we can make portable printers for skin. It is an adapted ink-jet-printer, to provide on-
site printing of skin for soldiers with life threatening burn. Here the skin cells are placed in the
sterilized ink cartridge, along with a material to support them. They are printed directly on the
wound.
The nano technology is used not only for printing the artificial organs but we can also use it to
preserve the tissues. Usually the organs are stored at 320° Fahrenheit. However, the organs
cannot be stored for long. For example, we can store the heart or lungs only for four hours
because they get damaged when you try to warm them up. So due to this more than 60% of the
donor hearts and lungs are not transplanted. But with this new method, the tissues can be re-
warmed with no sign of damage, and without any contamination.
In this method, scientists are using silica-coated iron oxide. This will be applied to the tissues
and an external magnetic field will be then activated, which will warm up the nanoparticles and
provide even heating throughout the tissues. Here none of the nano warmed tissues showed any
sign of damage, unlike the control tissues which was slowly reheated over ice. These
nanoparticles could be washed away during the process and it will prevent any contamination
associated issues, ensuring that this method is viable for tissue preservation.
Now let us see the organ de-cellularization approach. Instead of printing the complete organ, we
can use the de-cellularization approach and we can de-cellularize the organ from the donor. Let
us see this approach in detail.
We can have a customized organ using this approach. For example, if you want to construct a
new heart, we can remove all the cells from the donor heart leaving only the protein scaffold.
Then we can inject the cells and grow on top of this heart scaffold. We can then provide growth
factors and mechanical simulations and make the customized heart.
In the first step the detergents are pumped into the aorta, filling the arteries that feed the heart.
These detergents flow through the blood vessels and dissolve all the cells. Once the cells are
removed, you will be left only with the particular scaffold. Then precursor cells can be pumped
into the blood vessels and heart muscle precursor cells can injected into the muscle spaces. Once
the patient cells are grown on this particular scaffold, it can be transferred to the bio reactor. In
the bio reactor, a pulsing flow of nutrients will force the heart to beat. We can also give electrical
stimulations, which will help the heart muscles to contract on their own. Inside the bio reactor, it
will work like an exact heart. After monitoring in the bio reactor, we can transplant it into the
patient.
Recently, researchers have grown the heart tissue on a spinach leaf using the above approach.
The spinach leaf is stripped of its plant cells through a process called de-cellularization using
detergent, leaving behind the leaf’s vasculature. Then cells were cultured on top of these de-
cellularized leaves.
So using the spinach leaf vasculature as a scaffold, researchers have grown these beating human
heart cells on the de-cellularized leaves.
Let us see some of the benefits of organ printing. One of the major benefits is that we are going
to take the patient’s own cells. Hence the possibility of immuno rejection is removed. The organs
made by this approach will not wear out or need any occasional maintenance like the fully
mechanical organ transplants. The 3D printing technology will further eliminate the need for the
scaffolds and we can bypass the organ donor list. Another benefit is that using the computer
aided design, we can make customized organs which suits a particular patient.
Now let us see some of the difficulties and limitations in this organ printing technologies. So the
main thing is it is difficult to print vascularization in an organ, so effective blood flow in the
organ has been a major roadblock. The lifespan of the organs is also very limited. It can range
from few minutes to days. Hence the longevity of the organs need to be worked on before they
can be transplanted into the patient. Some organs have advanced functions beyond movement,
storage and filtration. For example, the liver’s ability to regenerate, which have not been
replicated in this lab made organs.
So the future goals are to develop more refined printers that can print even smaller details. This
will eliminate the need to make a separate vascular system for the organs. Also it can increase
the lifespan of the artificially printed organs. We should reduce the cost of this technology so
that it can be made available to more people.
So to summarize, in this lecture we have learnt what organ printing is and what the types of 3D
printing are. We have also learnt various 3D bio printing approaches. We have discussed about
the organ de-cellularization approach and also the role of nano technology in organ printing. So I
will end my lecture here. I thank you all for listening to this lecture. I will see you in another
interesting lecture.
For Further Details Contact

Coordinator, Educational Technology Cell
Indian Institute of Technology Roorkee
Roorkee – 247667
E Mail: etcell.iitrke@gmail.com. etcell@iitr.ernet.in
Website: www.iitr.ac.in/centers/ETC, www.nptel.ac.in
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INDIAN INSTITUTE OF TECHNOLOGY ROORKEE

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