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Abstract

Content

Introduction

Treatment is difficult to heal wounds, ulcers and burns – one of the areas of medicine, where for a long time and are widely used and continue to develop methods of cell therapy.

In 1975, it was proposed the use of cultured skin cells (keratinocytes) in the treatment of burns. Keratinocytes – the cells that make up the top, constantly updated layer of skin – the epidermis. The most commonly used autograft – that is the patient take a small piece of skin, it is isolated from keratinocytes grown in a culture and close formation received the wound surface. Transplants grown epidermal layers is one of the most complex and crucial stages of treatment technologies, on the correct response which often depend on the results of treatment.

Below are the main reasons for unsuccessful transplants epithelial sheets [3]:

  1. Lack of willingness to plastic layers. The optimal layer is composed of 8–12 layers of cells. Premature or late transplant ineffective.
  2. Transplant immature (young) formation. Typically, the surface of the culture flask uneven cells overgrow. Dispase treatment of a culture leads to uneven removal of the reservoir, with holes. The effectiveness of such low transplantation.
  3. Transplantation overripe cell layer. If you have many layers of cells breaks down food in the basal layer of keratinocytes.

One of the conditions of successful engraftment multilayered reservoir keratinocytes is their timely transplantation wounds. Regular monitoring of the viability of the cultured layer, achieving timely notice possible deviations and decide to maintain quality fragment formation or mattress seeding new culture. The problem is that the late release of notoriously poor quality mattresses cultures leads to the loss of significant financial resources, and most importantly time, which is often a decisive factor in the treatment of patients with extensive lesions of the skin. The amount of equipment for cultivation of keratinocytes is usually limited, and the material for transplantation, which can be done in store, is often necessary in large quantities and in a short time, such as during made disasters that are prevalent in our country.

1. Subject area

Quantitative output cells of the skin flaps and the effectiveness of their culture depends on:

  1. Storage duration flaps of skin from the moment of cutting before the cell isolation.
  2. Temperature at which skin biopsies contained during transport of clinic to the laboratory, where cell culture is carried out.
  3. Composition of the medium in which the specimens of the skin.

Temperature. In the case of storage at room temperature, skin grafts taken or from burnt skin donors allocate sufficient number of viable cells fails, keratinocyte proliferation in culture occurs sluggishly. When storing the pieces of leather refrigerated (at t = + 4 °C) the vital properties of the cells are retained for a long time (several days). It has been found that isolation of cells with in time to within 48 hours after cutting the skin keratinocyte viability decreases slightly and provides sufficient yield of viable cells.

Thus, the skin flaps should be stored at low temperature (from 0 °C to + 4 °C), should strive to ensure that the time interval between sampling prior to discharge cells was shorter.

Select transport medium. Besides temperature to affect cell viability and composition of the medium in which the transported cut skin grafts. In studies before Storage skin samples were thoroughly washed with Hank's solution with antibiotics and self storage performed at t = + 4 °C Dulbecco medium or 199 supplemented with 10% serum large cattle [4]. In the UK, for this purpose, DMEM medium supplemented with 5% fetal bovine serum and antibiotics.

To a much lesser extent for storage and transportation of the skin flaps are suitable salt solutions (in particular, Hank's solution). Using more simple solutions (glucose, 0.9% NaCl and others) does not allow for a long transportation time.

Thus, as a transport medium to use preparations used for cell culture and having a complete set of essential nutrients.

Number of allocated skin cells and their viability is largely dependent upon temperature storage and use of the composition of the transport medium.

Isolation keratinocytes. It is known that not all cells have the ability epidermis proliferation. Culturing makes sense to distinguish primarily cells the basal layer.

Cultivation of keratinocytes. Efficacy culturing keratinocytes and formation laminated reservoir depends upon a number of factors:

  1. State of the cells (viability and proliferative capacity).
  2. Composition of the growth medium used additives and growth factors.
  3. Type substrate on which the cells are seeded.
  4. Specific technology option cultivation.

Keratinocyte cultivation technology is extremely complex and can only be implemented in specialized biotechnology centers. At present we know quite a lot versions of this technology, differing composition of the medium, the presence of certain growth factors and additives.

Cell culture is carried out in the presence of feeder layer of transformed ZTZ fibroblasts pretreated with mitomycin C or exposed to sublethal doses in order to stop their proliferation. In this state, the cells were conditioned growth medium was biologically active substances that stimulate the growth of keratinocytes.

Keratinocytes cultured in Petri dishes, or in special flasks. Incubation of cells is performed incubated at 37 °C in an atmosphere containing 5% CO2. In case of correct isolating cells of the basal layer after 48 hours, the formation of colonies of keratinocytes. The growth medium was changed every 3–4 days. If necessary, carry cell reseeding culture.

Multilayer formation layers of keratinocytes. After seeding the resulting suspension into cell culture flasks keratinocytes attached and spread on the surface of the substrate, and then begin to actively share and form colonies. Last gradually coalesce and form a continuous layer keratinocytes. The rate of formation of the reservoir will depend on many factors:

  1. On the number of inoculated cells and their functional state.
  2. The composition of the culture medium and growth supplements used.
  3. On the method of preparation of the substrate, that is the processing of the bottom of the flask.

Formation multilayer formation during cultivation of the keratinocytes according to the method of Greene normally takes 3–4 weeks. Education cell layer occurs in the following sequence: attachment and cell spreading, increased division, colony formation and fusion of colonies together on the surface of the culture flask.

After attaching the unit cell colony formed quickly enough. Single cells also may be attached to the substrate surface. In this case, a relatively smooth cell monolayer. Colony by dividing cells increase in size and gradually merge. The central portion of the colonies resulting from differentiation becomes multilayer occurs stratification of the colony. After some time, a continuous multi‑layered (stratified) layer.

Determining the degree of readiness multilayered reservoir keratinocyte transplantation wounds. One of the conditions for successful engraftment multilayered reservoir is their timely transplantation wounds. For this it is necessary to determine the readiness of the formation of multilayer keratinocytes to transplant.

Young or immature layer thinner, has a weak cell‑cell communication and in the process dispase enzyme treatment may be destroyed. As stratification cell layer worsen conditions for absorption in the basal layer of keratinocytes. For this reason, the formation mnogomloyny perederzhivat can not, must be timely to assess the degree of maturity and implement transplantation.

On maturity cell culture judged mainly on the pattern observed in inverted microscope.

2. Statement of the problem

Important stage for a successful operation, is to determine the viability material.

Viability can be carried out by different methods. The simplest is adding special dyes that penetrate cells only when it is damaged shell. Thus, the stained cells were recognized not viable. Naturally, no dye is added to the whole material is obtained, but only in its pattern, followed by calculation of the level of cell viability of all abstracted.

More sophisticated techniques to evaluate the viability reduced to determine if cells carry out its functions [1]. For this primary culture are calculated levels specific substances that are produced by cells in the normal state. Reduction the concentrations of these compounds indicates that externally may appear intact cell functionally defective, that is not suitable for further use. Disadvantages these methods are time-consuming and rank as the subjectivity of the results.

Analyzing the existing methods for determining viability of cultured keratinocytes we can conclude that they have a material impact on the cells [8]. This requires regularly set aside a portion (albeit small) of crop. Thus, there task of developing a method that does not have a material impact on the cells.

3. Solution of the problem

To solve the problem to evaluate the viability of keratinocytes was chosen technique for handling them images since the use of this technique requires no physical effect on cells.

Inputs to the system being developed will be pictures with cell size mattress 512×512 pixels obtained using a camera mounted on the microscope. Sample images presented fig. 1.

Examples input snapshots

Figure 1 – Examples of input images
(animation: 5 frames, 6 cycles of repetition, 127 KB)

Next must be received its characteristic features, which can be used for classification. Preview keratinocyte layer has a complex texture. Also, there is the influence operator when taking still pictures as well as a microscope can be set to a different scale and rotate it from different angles. Therefore there is a need to make the image tilt and scale‑invariant. Similar problems were considered in [2], the study fibroblasts. To resolve this issue may be subjected to the original image log‑polar transformation.

Algorithm log‑polar image conversion keratinocytes divided into two main stages. In the first phase of the largest circle radius (r) within this image is used as a scanning line sample (sample) S times from 0° to 360° to get the equivalent S×[N/2] polar form (see fig. 2a) [5]. Thus, formally, the polar form p(a, r) of the image f(x, y) the size of N×N may be computed as:

Формула 1

for a = 0, …, S – 1, and r = 0, …, [N/2] – 1.

In the second stage logarithmic function applies to all values of radius in polar form, and their output values are quantized to R states. Obtain the log‑polar image size S×R for a given N×N image, as shown in fig. 2b.

Формула 2

for i = 0, …, S – 1, and j = 0, …, R – 1.

Log-polar transformation

Figure 2 – Schematic representation of the parameters to obtain the log‑polar transformation: (a) – get the parameters of the polar form p(a, r), (b) – get parameters log‑polar image

Further from the resulting log‑polar image is planned to extract informative features: specific energy and specific entropy. For this plan to use the wavelet transform [6, 7].

Conclusion

Analysis was conducted to evaluate the viability of existing methods of keratinocytes and justified method Imaging. The results of the research developed an algorithm imaging and determining informative signs to assess the viability of cultured keratinocytes. In the future we plan to conduct computer experiments with reference images keratinocytes to test the effectiveness of the selected method.

List of sources

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