Organ culture

Cell culture: Cells grown in monolayer on tissue culture dishes lack the three-dimensional (3D) cell–cell and cell–matrix contacts and communication present in intact tissue.

Although the general aspects of cell can be studied, the integrated function of the whole organism requires more complex models.

As a result a culture was developed in whichthe arrangement of cells resemble that in vivo.

Organ culture means the explantation and growth in vitro of organs or part of organs.

This type of tissue culture preserves the various tissue components their anatomical relationship and function in cultureso that the explanted tissue closely resembles its parent tissue in vivo.

It is to study the behaviour of integrated tissue, hence structural integrity is important

The in vitro conditions help to sustain life and function of the organ for a period of time. Hence, the cultured organs retain their physiological features.

What happens when organs are cultured?New growth seen•Glands: new glandular structures are formed.•Lung tissue: new small bronchi develop at the periphery of the explant..Retain their physiological features•Hormone-dependent tissues: remain hormone sensitive and responsive, and endocrine organs continue to secrete specific hormones. •Fetal tissues: morphogenesis in vitro closely resembles that seen in vivo.

However, outgrowth of isolated cells from periphery of explants is minimized by manipulating the culture conditions.

The idea behind organ culture is just to maintain it and not propagate it.It must remain viable and be functional.

Removal of organs:Are to be removed aseptically and first placed in BSS or HBSS supplemented with antibiotics. Strict aseptic conditions are needed to avoid contamination and to retain the organ structure and function.

To allow their growth:Grown on the solid support than in fluid medium. These are supplied with oxygen to prevent necrosis.

Checking the organ explants:The viability of the explants and macroscopic changes can be monitored during cultivation by viewing them by daylight or with the aid of a light source from the dissecting binocular.Healthy and growing tissues usually appear translucent with a shiny surface; opacity suggests loss of viability or the beginning of necrosis of the explanted tissue.

Types of organ culture•Plasma clots.•Agar substrate.•Raft method.•Grid method.•Cyclic exposure to medium and gas phase.

Plasma clot/solidified plasma

Watch glass technique( 1929)

Explant is cultured on the surface of plasma clot placed in watch glass (which may/maynot be closed with lid)

Prevents evaporation

Maintained at 37*C

Fresh medium to be supplied after 3-4 days

•Std classical tech for studying morphogenesis in

embryonic organ rudiments•Modified to study action of hormones, vitamins

and carcinogenes on adult mammalian tissues

Disadvantages

organ explant gets immersed in the medium.

Duration of culture is short hence, biochemical analysis not possible due to complexity of medium

Agar Gel

(1940)

The growth medium is gelled with 1% agar, kept in watch glass.

Avoids immersion of explants in the medium.

Subculturing required

Explants can be visualized with microscope

Application

To study developmental aspects of growth and differentiation of adult and embryonic tissues

Disadv: cannot grow for long time

Floating Raft method

(1954)

Explant kept on raft of lens paper or rayon acetate (treated with silicone) floated on the serum in a watch glass.

On raft ≥ 4 explants can be kept.

Prevents sinking of explants in the medium

Possible to study cell-cell interactions by growing different explants of different organs

Disadvantage

Floats often sank, tissues got immersed into medium.

Grid method

(1954)

This method utilizes wire mesh or metal grid whose edges are bent to form 4 legs.

Tissues directly placed on this (skeletal)

or on lens paper and then placed on the grid (soft tissues).

Placed in a chamber filled with fluid medium and supplied with mixture of 02 and CO2

Application:

To study growth and differentiation of adult and embryonic tissues.

Cyclic exposure to medium and gas phase

This method has been successfully used for long term culture of adult human tissues for 4-5 months

The explants are intermittently exposed to gas and liquid phase

The no. of explants per dish varies from 4-18.

The explants are cultured by placing them at the bottom of the plastic culture dish and are covered with fluid medium.

The dishes are enclosed in a chamber containing a suitable gas phase and mounted on a rocker platform. The chamber is rocked several cycles/min to ensure cyclic exposure of the organ explants to medium and gas phases.

Application:

Culture of human adult tissues such as esophagus, Mammary epithelium, uterine endocervix.

Advantages• Organ culture has a major advantage over primary cell cultures and cell lines in that most or all of the physiological properties of the organ are maintained.

• They remain comparable to in vivo organs both in structure and function

Since the hormone secreting organs release hormones, developmental stages in fetus are same as that of fetus in vivo.

Organ cultures provide information on patterns of growth, differentiation and overall development under the influence of different conditions.Considerably reduces cost that is incurred when whole animals are used

Disadvantages

• Organ cultures are difficult to prepare and maintain than cell cultures. The effects on these will appear only after several months using this model

• The throughput of organ culture is limited by the manipulations necessary, to remove the organ surgically from the host and set up the culture system.

• Production of explants has to be carefully done to maintain the structural integrity of tissue Only one, two or a few cultures can be obtained per donor animal.

• These cultures cannot be propagated. They can be maintained only for very few months.

• Reproducibility problems.

• The main problem is the gaseous diffusion and exchange of nutrients becomes limiting, which leads to necrosis.

Applications

• Studies on patterns of growth, differentiation and development of organ rudiments and influences of various factors like hormones, vitamins etc.

• To study the effect and action of drugs, carcinogens.

• Used as models for drug delivery and action.

• To produce organs for implantation in patients often called as tissue engineeringe.g. Human skin successfully cultured in vitro and used for transplantation in more than 500 cases of burns.

Explant has to come from patient himself, to avoid rejection. Not only burns treatment, but also skin defects such as chronic skin ulcers

Artificial cartilages to used for implantation for injuries and arthritis.

Organ cultures also are currently being used for studies of cardiovascular function,angiogenesis, thymus, skin, bone and urogenital tissues including kidney and bladder.

However organ culture is too slow and costly for drug screening and drug target screening,Though it has many other applications in biological research.

Embryo culture

Much of the organ culture has been performed on the embryos.

The organ rudiments can develop in vitro in a similar manner in vivo.

Intact embryos are of immense importance to understand the normal development, growth and differentiation and effect of toxic or teratogenic agents.

Human embryo development understood from animal models.

Can go for embryo removal or in vitro fertilization (fusion of ova and sperm in vitro)

•Human reproductive biology or to solve human infertility.

•In animal breeding

•Trangenic animals

Other culture methods which start with dispersed cells and encourage the arrangement of these cells into organ like structures.

Rather than preserving the organ structure, the 3-D structure is actually reconstructed in vitro.In these systems important physiological factors can be quantified thus overcoming some of the disadvantages of organ culture.

These can be sub-divided into :•Histotypic cultures•Organotypic cultures

Histotypic cultures:Culture systems using one characterized cell line propagated at high density in the presence of appropriate extracellular matrix and soluble factors are called as histotypic cultures

Uses only one cell type, hence Homologous cell interactions occur. Eg. Vascular endothelial cells form capillary tubules in the presence of appropriate soluble factors when grown in collagen matrix.

Organotypic cultures:When cells of different lineages recombined/re-aggregate to create tissue like structure the culture is called organotypic.There are heterologous cell interactions which are important in initiating and promoting differentiation.

Organotypic cultures are typically made by suspending stromal cells in growth media containing collagen I, which is liquid at 48*C.

Allowing the solution to harden into a gel at room temperature or above.

Epithelial cells then are layered on top of the gel and grown in a submerged state until a confluent monolayer is achieved.

These are placed at the surface of the liquid media and grown at the ‘air–liquid’ interface.

AdvantageReconstructed organotypic models are intermediate between monolayer cell cultures and in vivo models with regard to biological relevance, ease of manipulation and monitoring and cost.

DisadvantageOrganotypic cultures are limited by their lack of biological parameters such as immune and vascular systems. Blood vessels have been grown in organotypic culture, but their use has been limited to studying interactions of endothelial cells with fibroblasts.

ApplicationThe simplest type of organotypic culture is co-culture of two cell types.E.g. is co-culture of epithelial and fibroblast cell clones derived from mammary gland allows the epithelial cells to differentiate functionally.

1. Living skin equivalent (LSE) organotypic cultures The best-characterized reconstructed organotypic culture is the living skin equivalent developed in the 1980s and is still being used today.

Other application:Skin equivalents are currently being used to study antioxidant protection against photodamage, drug metabolism, differentiation , gene therapy and the immune system.