For example, organ transplantation is only treatment at the end stage of organ failure.

This procedure involves mainly remove the failed organ from the patient and replaced with the procured
organ for transplantation.

With today’s medical advancement, it can be done for any organ – liver, kidney, heart, etc.

But this whole procedure should follow legal and ethical considerations.

Organ transplantation society encourages the use of organs donated from the cadavers instead from the
living donors in order to reduce the risk for living donors while transplantation.

Although such therapies have merit to save the lives of thousands, shortage of donor organs and tissues as
the patient waiting list number has increased per year tremendously; and requirement of
immunosuppressant limits its potential to address organ failure crisis.

https://www.nytimes.com/2022/01/10/health/heart-transplant-pig-bennett.html
In case of surgical reconstruction, there is no possibility of immune rejection because of the use of patient’s own tissue
(autologous).

However, autologous grafting chiefly requires surgery at donor site; even limited supply, inadequate size and shape (Complaint
mismatch) with donor site morbidity restrict its use towards tissue loss.

In some cases of organ failure, say for example loss of hand; or loss of leg; patients are advised to use artificial prostheses. But
they are biologically non functional and they do not behave physiologically as a true organs.

Examples are artificial heart, heart valves, prosthetic hip, and artificial breast. But these materials are subject to fracture, wear,
toxicity, inflammation, which could induce the long term complications and rejections at the later stage.

Hence limitations of existing therapies provoke the search of new technologies or therapies as tissue engineering to
combat the organ failure and tissue lose crisis. Prof. Cato T. Laurencin defined Tissue Engineering as “the application of
biological, chemical, and engineering principles toward the repair, restoration, or regeneration of living tissues using
biomaterials, cells, and factors alone or in combination”.
 Tissue Engineering Triad

Biomaterials, cells and growth factors are known as the

“Tissue Engineering TRIAD”.

There are three different strategies that could be adopted for the regeneration of new tissues.

In the first approach cells can be used as therapeutic agents to restore the functional tissue. This approach mainly involves
the isolation of cells from different cell sources (autologous, allogenic; syngenic and xenogenic) and even use of stem cells
using a special technique called stem cell therapy, placing them in the site of interest for improving the tissue function.

The second approach involves the exogenous delivery of growth promoting substances like growth factors using the carriers
(polymeric or lipidemic) can stimulate the endogenous stem/progenitor cells for the specific differentiation thereby
replacing the lost cells or tissues.

The third approach is to use artificial 3-dimentional scaffolds or matrices for the growth of cells where cells can be either
recruited from the host tissue in vivo or seeded in vitro
Tissue engineering approaches
Even though these strategies can overcome the problems associated with the current clinical treatments, there are few
scientific challenges to construct a tissue or organ using these approaches.

For example,

use of cells: limited supply of autologous cells;

requirement of immunosuppressant for allogenic source;

disease transmission associated with the xenogenic source;

ethical considerations with the use of stem cells are the major challenges in case of first approach

Specific differentiation of stem cells with desired functionalities with the appropriate growth factor delivery is still unresolved
question in tissue engineering.

Even the third approach of tissue engineering is the development of artificial extracellular matrix (ECM) analogue towards the
regeneration of specific tissue.
 Challenges in tissue engineering

1. How do we develop a close approximate biological replica?

2. Should the tissue be produced in vitro implanted in vivo?

3. What type of scaffold should we choose?

4. How do we manufacture?

5. What cells are to be used?

6. Under what conditions can cells expand without affecting its phenotype?

7. What regulators do we use to stimulate the cell proliferation and

differentiation?
Hence to answer all the questions, it is necessary to understand the basic things such as

how tissue gets organized in the organ?

What are the different basic types of cells?

How do we classify the defects based on the tissues?

How well can a tissue repair its defects?

What are the components comprising the tissue

 Cells as Therapeutic Agents
Can we use cells as therapeutic agents to restore the tissue function?

Yes. Cells are the building blocks of tissues, which contribute to the respective functions.

Cell based therapies are a distinct class where cells are utilized as therapeutic agents to treat the pathological
conditions.

However, cell-based therapies are not new because for many decades people are practicing blood transfusion to
anemic patients to restore the oxygen supply.

Similarly, platelet transfusion for blood clotting defects;

bone marrow transplantation for cancer patients is widely practiced now.

Cell based therapies can also be used to repair the cartilage.

Hepatocytes and kidney cells are also used extracorporeal support devices to carryout the functions of the liver and kidney
respectively.

Similarly, beta-islet cells for diabetes,

sheets of fibroblasts for ulcers and burns,

genetically modified myocytes for muscular dystrophy have also been used clinically with varying degrees of success.
 Sources of Cells
The fundamental question is from where do we get the cell?
There are different cell sources available. They are

Autologous: Cell can be taken from the donor and introduced back to donor itself i.e., where donor and recipient are same.
Limited supply and the donor site surgery are the major drawbacks.

Allogenic: Cells can be transferred from donor to recipient of same species. However, difficult to reduce the patients risk
against the transplant antigens.

Syngeneic: Cells can be transplanted between genetically identical twins. Using this method, only twins can benefit but not
other living beings.

Xenogenic: Cells transplantation is between species. Where transmission of new diseases; and immune rejection concern
How do we use cells as therapeutic agent for various tissue defects?
we should first know what are the different types of tissue defects based on the functions?

Tissue defects can be classified as

mechanical,
metabolic,
synthetic,
communication and combination defects based on the tissue functions.
For example the major organ is affected in mechanical tissue defect is cartilage or bone since
the major function of these tissues are providing the mechanical strength to the body.

Similarly in case of metabolic defects, liver is the chief organ for metabolism. Hence is there
any abnormality in the liver function leads to metabolic defect.

Pancreas is the prime organ for synthesis of hormone like insulin, which is a primary regulator
for glucose metabolism. Thus any defect in the insulin production or dysfunction of pancreas
will lead to synthetic defects.

Nerve is the only organ used to communicate as well as coordinate all body functions. Hence,
the dysfunction of nerve due to any injury will promote the communication defects

Skin is largest organ performing multiple functions including immunological barrier; vitamin
metabolism; maintain homeostasis, and so on. Thus the defect in skin tissue is coming under
combination defects
 Cells used to restore the mechanical defects
Let us take an example of cartilage tissue defect.

First of all the major challenge in the functional cartilage restoration relies in the native
structure of this tissue.

That is, they are avascular (no blood vessels for nourishment), aneural (no innervations), and
alymphatic tissue (absence of lymphatic capillaries).

This tissue is an unusual biphasic tissue, which is made up of solid matrix called the extra
cellular matrix (ECM) and fluid phase (synovial fluid).

The major component of cartilage is ECM where terminally differentiated chondrocytes will
be dispersed at low densities. This architecture promotes frictionless surface with pain free
motion.

In aged population, osteoarthritis is the common problem where there is a deterioration of

cartilage including loss of chondrocytes and ECM architecture.
Cartilage defects in the knee can be treated by autologous transplantation.

Briefly, biopsy is collected outside the affected area from the patients and subjected to enzymatic treatment where the
chondrocytes will be harvested.

These chondrocytes are sub-cultured for expansion and then injected back to patient at the lesion. About 200,000 patients
are undergoing this type of cell based therapy every year

Autologous transplantation of chondrocytes in osteoarthritic patients

 Cells used to restore the metabolic defects
Let us take liver as an example. Liver plays a major role in metabolism
(protein, fats and carbohydrates), detoxification of foreign compounds,
production of vital serum proteins (albumin and clotting factors) and
production of bile for digestion.

Loss of liver function is mainly by hepatitis C, cirrhosis or excessive

alcoholism or even cancer.

Whole organ transplantation can use to treat liver defect at the end
stage.

However, this has many disadvantages like immune reactions/rejections,

limited supply of donor organs.

Hence there are other some temporary approaches can be used mainly
non-biological approach (Charcoal resins and dialysis) and biological
approach such as blood exchange and animal organ perfusion.

Extracorporeal hepatocyte based bioreactors are even used to culture

human hepatocytes and can able to maintain the liver function in the
form of bioartificial liver (BAL).
Hepatocytes are the chief cells present in the liver, which are highly proliferative cells.

But the major problem is associated with the hepatocyte in vitro culture is transdifferntiation, that is the
capacity of hepatocytes to differentiate into fibroblast.

Thus, the phenotype of hepatocytes is lost within a week and then it become functionally inactive.

Usually, a bioreactor with hollow fiber cartridge, charged with the immobilized hepatocytes (intraluminally or
extraluminally) is used.

This device is connected outside the body to blood or plasma circulation of patient.

The hepatocytes can also be embedded in a collagen hydrogel to avoid hydrodynamic damage and then
injected in the hollow fibers.

Due to the hydrogel nature of the collagen, it contracts thus forming a lumen, which helps in the diffusion of
medium and plasma.

This type of extracorporeal device can improve the detoxification and synthetic and even regulatory function
of diseased liver.
 Cells used to restore the synthetic defects
Pancreas is located behind the lower part of the stomach and the major clusters of cells in the pancreas is called
islets of Langerhans,

What is its function

It produce insulin and enzymes that help our body to digest food

In insulin dependent diabetic patients, pancreas loses its ability to secrete insulin. Allogenic cells are used (cells isolated from
the cadaverous pancreas), and injected into the liver portal vein thus enabling the pancreas to secrete insulin.

However, since the source of islet cells is allogenic, there will be immediate rejection due to recurring autoimmunity,
insufficient transplanted β-cell mass and inflammation.

Even the use of immunosuppressive agents provokes toxicity.

Similarly, in case of xenogenic islet transplantation, porcine islets are used since its glycemic regulation is similar to humans.

However, complications such as long-term survival of porcine grafts, lack of biocompatibility, poor immune-protective
properties, hypoxia and chances of disease transmission could limit the use.
Hence, physical separation of donor’s cells from the immune
system is mandatory to avoid immune rejection.

This could be achieved by the encapsulation of donor cells

using semi permeable membrane, which selectively allow the
passage of nutrients and insulin while preventing the passage
of antibodies and other biomolecules that could damage the
implanted cells as shown in the cartoon
 Cells used to restore the communication defects
How do we use neural cells to restore the function of nerves?

Nerve injury creates a gap or cavity due to the loss of myelin

sheath, thereby loss of impulse transmission.

Regenerating nerve, especially in the central nervous system is

really challenging since in the native environment axons in the
central nervous system do not regenerate substantially due to
the surrounding inhibitory environment.

Hence, major aims of the cellular therapeutic interventions in

the restoration of communication defect are:

i. To bridge the cavities

ii. To replace the dead cells and
iii. To create a permissible environment for axon regeneration
Cells such as Schwann cells, olfactory enshealthing cells can be used for achieve the above. The advantage of using
Schwann cells is mainly because it can produce ECM, cell adhesion molecules, and neurotrophins that lead the peripheral
axon to distal stump and synapse formation.

However, complications such as unfavourable interaction with glial scar components in CNS, inability to remyelinate the
axon beyond the injury is leading to be big challenge. In addition, Schwann cells are known to exacerbate the chondroitin
sulphate production, which hinders the regeneration further in astrocyte rich region. Hence, this can be replaced with the
use of olfactory ensheathing cells because of its structural similarity to Schwann cells and astrocytes.

Use of olfactory ensheathing cells can produce neurotrophins and cell adhesion molecules and migrate through the
growing axons and induce the sprouting of axonal outgrowth.

Hence, this provides a permissive environment for axonal growth and effectively supports the axonal outgrowth through
glial scars.

But again while harvesting the cells from the olfactory bulb one should ready to compromise the host’s sense of smell
 Cells used to restore the combination defects
Skin is the very good example for multifunctional
organ.

Skin is one of the most prolific tissues in the

human body. It consists of two layers dermis
consisting of fibroblasts and the epidermis
consisting of keratinocytes.

Variety of skin ulcers and burns are treated by the

transplantation of fibroblasts and keratinocytes
with no immune rejection.

This therapy is almost well developed and lots of

clinical products are now commercially available.
Normally ability of the skin to heal itself is
noteworthy but when the injury is very severe,
medical interference is essential to improve the
healing process. Also treatment strategies should
protect the wound area from the infectious
agents and limit fluid loss in the intervening time.
Burns

A first-degree burn wound which only affects the epidermis heals spontaneously, and without a scar.
Reconstitution of the epidermal integrity then results from the migration of keratinocytes originating from the
nearby epidermis or appendages.

A second-degree burn wound, defined as superficial or deep on the depth of the dermal burn, destroys the
epidermis and part of the dermis. It heals spontaneously by the migration of cells originating from the epidermal
appendages. However, a permanent scar will form.

A third-degree burn destroys the entire thickness of the skin (full-thickness burn). Although a tiny third-degree burn
wound can possibly heal with a scar by migration of cells from the surrounding unwounded skin, a large one will
not.

The transplantation of split-thickness autografts obtained from unburned donor areas is then the unique
therapeutic option, with the added risk of improper healing of donor sites, including infection, hypertrophic scars,
and cheloids.

The transplantation of autologous epidermal stem cells then becomes life-saving for those patients who do not
have enough donor skin for conventional split thickness autografts.
Methodology

A full thickness skin biopsy is obtained aseptically from normal skin, to isolate stem cells from both the epidermis
and the epidermal appendages.
The biopsy is then stored in culture medium and transferred to the laboratory, where it is minced with scissors and
treated with trypsin to obtain a single-cell suspension.
The cells are seeded at a density of 2.4-3x104 cells/cm2 onto a feeder layer of irradiated 3T3 cells in medium
containing fetal bovine serum, and supplemented with cholera toxin, hydrocortisone, insulin, and triodothyronine,
according to published protocols.
Cultures are supplemented with epidermal growth factor (10 ng/ml) at the first medium change. Under these
conditions, clonogenic keratinocytes divide every 16 to 18 hours, and colonies quickly expand and push away the
irradiated 3T3 cells, which then detach from the culture vessel. If the culture lasts long enough, the colonies of
keratinocytes will fuse and form a stratified epithelium.
Confluent cultured epithelia then need to be detached enzymatically from the bottom of the culture vessels, using
dispase or thermolysine. However, the enzymatic treatment significantly shrinks the cultured epithelium. Once
rinsed, each epithelial graft is mounted onto petroleum gauze backing, with special care being taken to maintain
the correct orientation of the epithelium, (i.e., the basal layer facing away from the backing). This step of the
procedure is cumbersome, tedious, and manpower demanding, especially when hundreds of cultured epidermal
autografts (CEAs) need to be prepared in a reasonable amount of time. This is why many groups have explored
alternative methods to cultivating keratinocytes.
 Cell numbers and growth rate
The cell density in the human tissue is in order of 1-3 billion cells per mL, hence
human body consists of about 100 trillion cells.

But if you consider an organ, it consists of few hundred million functional subunits.

The number of cells is determined by the functional ability of each subunit.

For example in kidney the number of nephrons (functional subunit) is determined

by the maximal clearance of toxic byproducts and also the clearance ability of each
nephron.

For cell-based therapies certain number of cells is required and it varies depending
on the type of tissue that is affected.
For example, chondrocyte transplants require few tens of millions of cells;

lymphocyte therapies require half a billion of cells,

liver support requires 10 billion hepatocytes and

bone marrow transplantation requires few billion cells.

There are certain limitations in the production of primary cells.

A single cell can produce 1010 to 1015 cells in culture. However, this depends on the type of the cell also.

Because fibroblasts and skeletal myocytes grow well in culture while hepatocytes and beta islet cells do not.

Usually primary human cells can undergo 30 to 50 doublings in culture based on the age of the donor and the
doubling is determined by the Hayflick limit.

Primary cells vary in growth rate.

For example, the doubling time of hematopoietic cells are 11-12 hours while dermal fibroblasts require 15 hours.
Adult chondrocytes require 24-48 hours for doubling while hepatocytes do not grow in culture at all.

Cells are cryopreserved with suitable cryoprotectants like glycerol and dimethylsulphoxide. However, solution
effects have to be taken care to avoid intracellular ice formation during freezing. Viability of cells after the
cryopreservation process may vary from 40% to 95%. For treatment the cells are usually injected into the portal
vein or it can be encapsulated in a suitable polymeric scaffold and then delivered to the patient
Supplementary notes
 Vegf = Vascular endothelial growth factor
Monocytes - macrophages PHAGOCYTOSIS Neurons
Astrocyes
LPS Glial cells
Inflammation
Microglial cells
Engulphin foreign substance Oligodendrite cells
Many other immne cells
Bacteria Monocytes
Neutrophils
Eosinophils
Lymphocytes
Basophils
fUSOSOME Acidi Macrophages
c
Macrophage NK cells

Lytic Enzymes
T CELLS
B lymp

Endocytosis Tc
eXOCYTOSIS
Th Cytokines B Cells
MHC - Antigens
Plasma
Abs
Polymers
DNA Polymer of nucleic acid - Phosphodiester bond

Chain

Starch carbohydrate – glucose and galactose - WATER

Cellulose – Water

Chitin – Chitosan – soluble in acidified water

Colloid or emulsion == lipid –mix –water

Oil –mix-water = O/W emulsion VEGF is a protein – Water
Oil

Mix

W/o emulsion
Oedema --- MRI--- Water relaxivity - Tesla
 Neural Cells

Stem cells

Fibroblast cell also

NGF