Tissue decellularization

Tissue decellularization is a techniques which aims the substution of artificial scaffolds by decellularized organs. Decellularized tissues have several advantages with respect to the man-made scaffolds since they reproduce much more accurately the structure of the extracellular matrix in which cells live in-vivo, and may even incorporate some of the chemical and mechanical signals which lead the cells to behave in a determined 'realistic' way.

However, several considerations must be taken into account when dealing with these tissue-based scaffolds. The first one is that before being used as scaffolds, all the cells and biological rests of the original tissue content must be completely removed. This process is knows as "decellularization" and is usually accomplished by circulating detergent-based solutions through the organ walls. Those solutions destroy and remove all the original living cells without affecting the ECM tissue which, in the end, results in an 'empty' matrix tissue.

Although conceived to develop culture under flow conditions, the EBERS tubular chambers can be straightforwardly used for decellularization experiments just by replacing the culture media by a detergent solution.

 

Decellularization process

 
 

Organ harvesting

First step in the decellularization process is harvesting a tissue or organ from a donor.

 

Experimental set-up configuration

Next, the the organ is inserted in the Static Tubular Chamber. Both luminal and the external circuits are used to make the detergent solution go through the sample.

The color of the organ starts turning from red to white as the remaining blood and components are taken away from the extracellular matrix.

 

Decellularized tissue

When the decullularization process is finished, the tissue present a white color corresponding to the reamining protein structures composing the ECM.

 

Recellularization

Once all the original cells are removed from the tissue, the sample is washed and is ready to be recellularized. The Static Tubular Chamber can be used again for this purpose just by replacing the detergent medium by culture medium with a high cell concentration solution. As the medium is pumped through the scaffols walls, cell get attached to it.

Cell culture on plane surfaces

Maybe the simplest application of flow to cell culture is a experimental set-up for cell grow on a bi-dimensional surface. Although simple, this experimental set-up is also highly versatile and has been intensively used to study the effect of shear stress caused by culture medium flow on cell functions.

EBERS TEB series bioreactors easily allow culturing cells on bi-dimensional surfaces subjected to flow conditions. As an example, the main components of a suitable experimental set-up are shown below.

 

Materials and methods

 
 

A feasible 2D culture plaque

Both EBERS bespoke chambers and third party chambers can be used for the set-up. A commercial disposable 2D culture chamber is used in this case.

 

Experimental set-up configuration

The experimental set-up configuration determines the number of samples which can be grown simultaneously. Setting up several culture plaques in parallel may be an excellent way to increase the productivity of your experiments with a fixed culture conditions.

Only of the two independent pumping systems available in the TEB1000 bioreactor equiped with a one channel pumphead have been used in this set-up.Then, in order to increase the number of simultaneous experiments, an appropriate tube distribution has been used.

 

Tube connection detail

Tube connexion must be carefully done in order to guarantee the appropriate flow conditions on the cultures. Then, the only remaining step to start with the culture is to program the TEB1000 so the desired flow is pumped through the chambers.

Three-dimensional cell culture

Obtaining an homogeneous cell growth in scaffolds becomes extremely difficult when scaffold are thicker than 2-3 mm. This phenomenon, which has been experimentally observed in a multitude of studies, is causes by the lack of nutrients and oxygen in the internal zones of the scaffold caused by cellular metabolic activity.

A feasible solution to this situation is based on the establishment of culture medium flow through the cultured scaffold (perfusion). By means of this technique the amount of nutrients and oxygen supplied to the cell living in the inner parts of the scaffold is improved, which is crucial for the development of those populations.

The EBERS TEB series bioreactors and P3D chambers are specifically designed to develop cell cultures on 3D scaffolds under flow conditions, e.g., under perfusion loads.

  

Materials and methods

 

3D cell culture

Cells can be grown on three-dimensional scaffolds in a wide number of ways. Using EBERS culture packages is a possible solution, while developing your own culture circuit might be the best option to satisfy the particular requirements of your experimental set-up.

 

Perfusion flow

Perfusion flow is crucial for the appropriate development of cells in thick scaffolds. EBERS perfusion chambers has been developed for that purpose and is suitable for different scaffold types and sizes as well as for multiple simultaneous experiments.

Multiple simultaneous scaffold culture. Improved scalability

Increasing the number of simultaneous experiments may be of crucial importance in those cases in which the scalability of the experiment plays an important role in the obtaining of the desired results.

EBERS bioreactors and chambers allow increasing the number of simultaneous in several different ways. The list below shows some of the feasible configurations that allow developing multiple simultaneous experiments.

 

 

Alternatives to increase the number of simultaneous experiments

 

 

Use each pump for a different experiment

Configuring two experiments in the TEB1000 or TEB500 bioreactors taking advantage of the two pumping systems is the easiest way to carry out two simultaneous experiments.

Moreover, since each of the pumps can be controlled individually, this configuration allow developing two totally different experiments. Nevertheless, this solution admits only two simultaneous experiments, which is not much in principle.

 

Using multiple pumpheads in each engine

For those applications which need of using the two pumps at the same time, connecting several pumpheads to the same engine may be a feasible solution. Until four pumpheads may be connected to each engine, which allows to multiply the number of simultaneous experiments. This possibility is only available in the TEB1000 bioreactor.

Notice, however, that the flow condition will be identical for those pumpheads connected to the same engine.

 

Using one or more multichannel pumpheads

The TEB500 bioreactor is equipped with two four channels pumphead by default, which sums a total of 8 channels. In which the TEB1000 is concerned, it allows using the Low Flow pumphead, which is a five channel pumphead. Moreover, up to four pumphead can be used at the same time in the TEB1000, which renders a total of 20 channels.

 

Using flow distributing devices

In case only a pumphead is used, including a flow distributor device in the circuit may also be a feasible solution to get multiple simultaneous connections. Generally, this devices are custom manufactured to cover the particular demands of each experiment.

 
 

Contact our custom development department for additional information on this devices.

Dynamic scaffold seeding via perfusion flow

The appropriate seeding of scaffolds is crucial for the good performance of the experiments. Recent experiments have shown that dynamic seeding using perfusion flow is an excellent method to get homogeneous seeded cell populations (see, e.g., the work Dynamic Cell Seeding of Polymer Scaffolds for Cartilage Tissue Engineering).

The EBERS TEB1000 bioreactor, the P3D chambers and the Seeding Rack can be used to develop dynamic cell seeding on porous scaffolds by configuring the experimental set-up shown below.

 

Materials and methods

 

Experimental set-up

Having a flow bioreactor lets you to easily configure a culture circuit for carrying our dynamic flow seeding of scaffolds.

For that purpose, it is only necessary to use a single pump to impulse the culture medium from two reservoirs are connected to the seeding chamber. Then, the high concentration culture media is pumped in alternative directions through the scaffolds thickness with the desired frequency so cells can adhere to the scaffold walls.

 

Flow profile

Regarding the flow profile necessary to recirculate the fluid through the scaffold, it can be easily applied in the flow bioreactor. Thereby, the high cell concentration media is recirculated through a scaffold so cells are seeded on it.

In addition, the seeding media should not go through the pumphead rollers, since they might squash cells floating in the medium.

 
 

Watch the main steps of the seeding process in this video!!!!
 

Measuring the permeability of porous scaffolds

The permeability of a three-dimensional biomaterial is a crucial parameter in order to determine the feasibility of such material to be used to manufacture tissue engineering scaffolds.

In general, the permeability of the scaffold has a strong influence on several aspects:

  • Nutrients and oxygen supply

    The permeability of a material is closely related to the suitability of the scaffold to host cells since it is related to the microstructure and determines how easy or difficult is to get nutrients and oxygen for those cell living inside the scaffold. Intuitively, nutrient and O2 supply will be more difficult in those scaffolds with a very low permeability.

  • Viability of long-term cultures

    The permeability of a scaffold is a parameter which may be nicely used to estimate cell development in a long-term experiment. In this sort of experiments, cells are cultivated for long periods of time aiming them to develop and to remodel the environment by generating the extra-cellular matrix that composes the desired engineered tissue as, e.g.. osseous matrix.

    Obviously, cell proliferation and matrix generation have a very important effect on the original scaffold porosity since matter deposition usually leads to a decrease in the overall scaffold permeability. Therefore, an excessive cell proliferation may cause the pore occlusion and the interruption of the supplies to the cell in the inner part of the scaffold.

  • Scaffold porosity under deformations states

    All the tissues are subject to mechanical loads in-vivo and, therefore, scaffolds on which cells are grown should also be ready to hosts living cell populations under mechanical loads causing them noticeable deformations. A typical example consists of a porous scaffold for osseous cells culture which, once implanted, should stand for the loads associated to, e.g., normal gait.

    Deformations associated to those loads may have a strong influence on the scaffold micro-structure, since pores shape and size might be modified causing a reduction of the scaffold permeability. Thus, the measurement of permeability in deformed states is a nice tool in the study of the suitability of scaffolds under noticeable deformations.

These reasons make of porosity an important parameter, frequently provided by the scaffold manufacturers, whose effect of the cell populations have been deeply studied in the literature.

The EBERS TEB series in combinations with the P3D chambers allow determining the permeability of a porous scaffold by means of a simple experiment. A feasible experimental set-up feasible for this purpose is shown below.

 

Set-up for scaffold permeability measurement

The experimental set-up depicted on the right side may be used to measure the permeability of a scaffold subjected to perfusion flow.

The physical principle used for this purposed is based on the measurement of the pressure drop caused by the introduction of the scaffolds in the culture medium flow. Thereby, the scaffold permeability can be easily determined by applying simple mathematical relations.

Adding a pressure sensor to control the pressure drop in the culture chamber allows estimating the permeability of the scaffold is the only additional equipment necessary for this experiment.