The working of diffusion cell apparatus or Franz cell is based on ‘in vitro diffusion.’ It consists of two chambers, a donor compartment and a receptor compartment, separated by a membrane. The product to be tested is introduced through the donor compartment (the top chamber). The bottom chamber or the receptor compartment contains fluids from which samples to be analyzed are extracted at regular intervals. The sample collected determines the amount of “active” that permeates through the membrane at point time. The cell temperature constantly remains at 37oC. The receptor compartment of a diffusion cell or Franz cell has a fixed volume, and it allows the stirring of both receptor and donor chambers (Kelin et al., 2018).

A diffusion cell apparatus helps to evaluate compound uptake into the membrane and determines tissue/membrane permeability, concentration in the receptor compartment, and flux. When working with a highly permeable compound and a large receptor compartment, the reduction in the gradient builds up the compound in the receptor chamber. On the other hand, if the receptor chamber is small, the concentration gradient is reduced due to compound buildup. The reduction in concentration gradient ultimately slows down the compound flux resulting in non-sink conditions. However, if the compound has low permeability, the detection of the compound in a large volume receptor chamber can be problematic.

Membrane Types

Various types of membranes can be used for diffusion experiments. Some of these are given below:

• Human membranes: Healthy human tissues can be used for permeability experiments on a large scale. Some laboratories also use human cadavers’ skin obtained from accredited tissue banks. However, there are some ethical issues with using healthy human tissues.

• Animals: Small and large animal tissues are used for diffusion experiments. Small animals like rodents are used for permeability experiments as they are inexpensive and easy to keep; however, they are morphologically different from human tissues and are relatively thin. Large animals like dogs, monkeys, pigs, etc., might also be used, as their tissue morphology is like that of humans; however, they are expensive.
• Synthetic/Polymeric Membranes:  Polymeric membranes are frequently used for in vitro release testing. They are synthetic, inert, and commercially available as cellulose, polysulfone, etc. Hydrophilic membranes with 0.45µm pore size are usually used for In vitro release tests (IVRT). Once set in the developing phase, the same membrane should be used for the entire experiment.
• Human Skin Equivalents (HSEs): These are synthetically engineered 3D human skin tissues prepared from a combination of human skin cells and extracellular matrix components under controlled conditions. Their main advantage is that they remain viable. However, they have higher permeability as compared to the tissues in vivo. Artificial membranes are beneficial as one can use them to predict the in vivo effect of compounds and formulations.

While selecting a suitable membrane, one must consider its source, integrity, time of excision, and shelf life. If it is obtained from an animal source, the scientist must know the health and age of the animal, and if the source is human, the researcher must consider the race, age, and gender of the human. If the permeability of the membrane/tissue is altered, it may affect the results of your experiment. Therefore, the experimenter must know beforehand whether the membrane was exposed to any chemicals. Skin tissue, however, may be refrigerated and used later without a change in permeability.

Donor Compounds

The donor compound can be prepared as liquid, gel, suspension, ointment, lotion, powder, or adhesive patches. One can determine permeant behavior and characteristics based on concentration and permeability.

1. Permeant Concentration

The permeant concentration depends on the aim of the study. An infinite dose is when the permeant does not deplete from the donor compound throughout the experiment and is used to test fundamental permeation behavior in the presence of permeability enhancers. On the contrary, a finite dose relates to the permeant amount that would be practical in actual usage, for example, a specific amount of drug to be administered to a patient. It aims to determine the amount of permeant required to be incorporated in a vehicle that will deliver the required amount of drug to the patient. Alternatively, the application of a compound that will temporarily remain on the tissue can be mimicked. In this case, the permeant is applied to the membrane for a short time and then removed. The researcher should then collect the samples for a given length of time following permeation.

2. Vehicle Formulation

The vehicle is the medium in which the permeant of interest is incorporated. Vehicle formulation is the “dose to be administered without the drug or active pharmaceutical agent (API).” Aqueous solutions and phosphate buffers serve as excellent vehicles for getting basic permeation data. Lipophilic compounds, however, have low solubility in aqueous solutions. Therefore, vehicles like water, alcohol, or propylene glycol should be used while working with them. Inert materials may alter the flux of compounds.

Moreover, the potency of enhancers may be altered by the nature of the vehicle. A selection buffer controls the degree of ionization; therefore, adjusting pH can completely dissociate the permeant into ions. Furthermore, vehicle components may also be modified by thermodynamic activity.

3. Permeant Detection

Spectroscopy and chromatography techniques are used for permeant detection.

4. Other Considerations

A group of five to six replicate doses should be administered to avoid errors resulting from the biological variability of skin. A minimum of three parallel cells should be used for testing each formulation.

Receptor Media

The receptor solution is selected based on the nature of the permeant. An ideal receptor compound should mimic in vivo situations. The accumulation in the receptor compartment is minimized by using flow-thru systems, and aqueous receptor fluid is sufficient for this purpose. On the other hand, it raises a concern for static cells where the permeant is not continually cleared. The solubility of the compound is a major concern in this regard. It should be such that it is in its desired form in the donor media and delivered in a certain amount to the receptor compound. Hydrophilic and lipophilic permeants show the best results with the aqueous receptors.

Similarly, PBS is used for ionizable permeants. If the permeant is un-ionizable, a solubilizer should be added to the receptor compound. Moreover, permeants with low aqueous solubility, like lipophilic permeants, require additional solubilizing agents such as surfactants or organic solvents. However, these agents might alter the membrane permeability and damage or alter the tissue/membrane. Therefore, one should use the minimum possible amount of these agents.

Sampling

The method of sampling depends on your research question. One can measure the amount of compound passing through the membrane at short intervals or the total amount that passes over a long-time duration. One can also determine flux, permeability constant (Kp), accumulation, etc., using a diffusion cell apparatus.

The researchers can use radiolabeled or unlabeled compounds for measuring flux and permeability. The results are reported as one of the two types of accumulation:

1. The total amount of the compound in the collected sample per time interval (irrespective of volume).
2. The compound concentration.

Radiolabeled compounds can be detected in very small amounts in large volumes of receptor media; however, the results are accurate and robust for such compounds. On the other hand, non-radiolabeled compounds used in permeability research can be detected using HPLC and ELISA methods.

Calculating flux and Permeability Constant

Flux is the amount of permeant passing through the membrane in unit time. It is measured in ‘Joules’ (J), and its formula is given by: J=Q/ (A x t). In the given formula, Q is the amount of permeant crossing the membrane in time t, and A is the membrane area usually given in cm2.

Steady State Flux is the amount of permeant crossing the membrane at a constant rate. This state is achieved after the lag phase as the amount of permeant continues to increase. When you measure the amount at regular short intervals at this stage, no significant difference in the values is observed. The units of steady-state flux (Jss) are quantity/(cm2*h). the formula for calculating steady-state flux is J = dQ/dt*A, where it is the permeation time, and A is the membrane area in cm2.

Permeability constant (Kp) can be calculated by using the formula Kp= Q/ [A*t*(Co-Ci)]. It is measured when an infinite dose of permeant is applied to the membrane. In the given formula, Q is the quantity of the permeant passing through the membrane in time t, A is the area of the membrane (cm2), Co is the concentration of the compound on the outer side (donor chamber), and Ci is the compound concentration on the inner side (receptor chamber).

The apparent permeability of the solution can be calculated by normalizing ‘J’ over drug concentration in the donor compartment Co, i.e., Papp = J/ Co.