RU2793462C1 - Method of cell-engineering construct sample preparation based on polylactic acid and eukaryotic cell culture for histological analysis - Google Patents

Abstract

FIELD: cell biology.

SUBSTANCE: invention represents a method of a cell engineering construct (CEC) sample preparation based on polylactic acid (LAA) and eukaryotic cell culture for histological analysis and can be used in regenerative medicine. To implement the method, first slides are preliminarily prepared, on each of which a polylysine layer of 250 μl of polylysine at a concentration of 1 mg/ml is evenly applied on top, and dried for 24 hours at a temperature of 37°C. After that, the sample of the cell-engineering construct is pre-contrast stained with 1% eosin solution. Then it is shock-frozen in liquid nitrogen at a temperature of minus 196°C. Then it is fixed in a cryomicrotome using a cryogel. Then, a cryomicroscopic cutting of the area of the cell-engineering structure is carried out, after which it is fixed on the prepared adhesive glass slide.

EFFECT: invention makes it possible to obtain CECs while maintaining the structure of the scaffold and the integrity of intracellular structures, which makes it possible to evaluate the interaction of the polymer carrier and the cell culture, as well as the proliferation of the latter.

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Description

The invention relates to biology, namely to histology, and can be used in regenerative medicine in the development of cell engineering structures (CECs) based on a polylactic acid (PLA) polymer scaffold and a eukaryotic cell culture during in vitro and in vivo experiments.

Currently, tissue engineering is being actively developed to replace defects in tissues and organs, which makes it possible to replace the damaged area with a cell-engineered structure based on a biodegradable polymer scaffold and cell culture, thereby restoring the functionality of this area. Biodegradable polylactide is often used as a polymer carrier, which is currently considered the "gold standard" of scaffold due to its safety for the recipient's body, biodegradability, and the ability to reproduce the specified characteristics of the 3D internal structure, which ensures the maintenance of viability and proliferation of cell culture. One of the advantages of polylactide is its economic availability.

Cell-engineered constructs (CECs) are used in limited clinical practice. To improve the characteristics of CIC, extensive experimental studies are currently being carried out in vivo on laboratory animals, as described in the works of Fioretta E., von Boehmer L., Motta S., Lintas V., Hoerstrup S., Emmert M. [1] and Li Ming, Yuan Zhipeng, Yu Fei, Rao Feng, Weng Jian, Jiang Baoguo, Wen Yongqiang, Zhang Peixun. [2]. The main goal of in vivo experiments is to evaluate the responses of perifocal tissues to CEC implantation and scaffold biodegradation. For this, clinical (assessment of mobility in the joint), radiation (magnetic resonance imaging) and microscopic research methods (scanning electron microscopy, histology) are used.

The most widely used is the classical histological examination with the posting of the studied tissue blocks for various reagents, the manufacture of paraffin blocks and subsequent deparaffinization before staining, including the use of histochemical techniques. Studies of the defect area using histological methods make it possible to assess the biocompatibility of the developed CEC with surrounding tissues and to study the dynamics of perifocal tissue reactions.

Before conducting an experiment on laboratory animals, in vitro studies are carried out. Microscopy of CEC preparations makes it possible to evaluate the characteristics of the resulting biomedical cell product, including studying the depth of scaffold occupancy, the number and viability of cells. However, the well-known classical histological methods in some cases are not suitable for the analysis of CICs based on polymer carriers - both in vitro and in vivo , since the polymer carrier can degrade and dissolve during histological sample preparation, which is described in the methods of Teixeira, S., Eblagon , KM, Miranda, F., Pereira, MFR, Figueiredo [3] and I. -Franco, F., Auras, R., Burgess, G., Holmes, D., Fang, X., Rubino, M., Soto-Valdez H. [4].

In addition to the classical histological approach associated with the process of manufacturing paraffin blocks and subsequent deparaffinization, a technique based on obtaining cryosections at low temperatures is used. At present, the cryosectioning method is widely used to study CFCs.

One of the important properties for the successful production of cryosections is the adhesive ability of the glass slide. This is due to the fact that in the process of staining the histological cryosection, it is necessary to exclude the detachment of the sample from the glass surface. For these purposes, specialized glass slides coated with polylysine are used. However, even in this case, the production of CIC cryosections requires special skills, selection of parameters, and specialized glasses.

An analogue of our methodology is the sequence of actions described in the Brekke, John H. (Duluth, MN) patent [5]. This technique uses only scanning electron microscopy and does not allow obtaining data on the inner layers of the regenerate area and the created CIC. Despite the ease of use and the guaranteed effectiveness of obtaining research objects, this method has significant limitations. The presented method cannot answer the question about the belonging of individual cellular elements. The technique described in the patent cannot be used to analyze the effectiveness of CEC, since it does not present the methodology for preparing histological preparations, but only the method of scanning electron microscopy (SEM) is indicated.

Fundamentally different is the technique described in the patent (Estes, Bradley T. (Durham, NC, US) Moutos, Franklin Thomas (Raleigh, NC, US) Guilak, Farshid (Durham, NC, US), [6]. description of the preparation of classical histological preparations.The presented method allows obtaining histological sections of the regenerated area and the created CFC by creating classical paraffin blocks, subsequent deparaffinization and histological posting of sections.However, the sequence of actions described in this method does not provide specific parameters for the preparation of histological preparations, the manufacture of blocks and coloring techniques.

A method is known that includes classical histology and describes the specific steps in the preparation of histological sections, which was developed in 2013. This technique, proposed by a group of researchers led by James W Edinger [7], also uses sequential preparation of histological preparations with the preparation of paraffin blocks and subsequent deparaffinization, cutting and staining of histological sections. The advantage of this technique is the possibility of histochemical staining of the tissue section with CIC. Significant disadvantages of this technique are: the possibility of carrier degradation during deparaffinization and the lack of an accurate description of the polymer scaffold; the possibility of obtaining micropreparations of CEC sections before and after implantation is not shown.

Some of the shortcomings of the above methods were eliminated in the work of Pathak, Chandrashekhar P. and Thigle, Sanjay M. [6]. In the method proposed by the authors, a fundamentally similar method of standard histological wiring is described in detail and at the same time the theoretical possibility of partial degradation of the polymer carrier is indicated. An additional advantage of this technique is the simultaneous shock freezing of the scaffold. However, the proposed method also has significant drawbacks. For example, the authors did not indicate the possibility of obtaining micropreparations of native scaffold sections or finished CIC without implantation in animals. An additional disadvantage is the use of alcohols in histological wiring, which disrupt the structure of the polymer and (or) the whole CIC. Separately, it should be noted the difficulty of fixing the polymer section with cells on the surface of the glass slide, which creates difficulties at the stage of staining the histological section.

Among the methods for CFC analysis, one can single out a group of methods that differ in an important feature, which was described in the work of Morille, Marie, Tran, Van-thanh, Garric, Xavier, Coudane, Jean, Venier-julienne, Marie-claire, Montero-menei, Claudia [9] and Sehl, Louis C., Trollsas, Olof Mikael , Mallace, Donald G., Toman, David, Delustro, Frank A., Schroeder, Jacqueline A., Chu, George H [10]. These techniques include the use of a single-layer adhesive base (polylysine) to improve the fixation of the histological section on the slide during staining. Another important feature of this technique is the partial use of scaffold freezing for further analysis. However, despite these advantages, the methods also have significant drawbacks. The main one is the need to obtain classical paraffin blocks followed by dewaxing and coloring, which can lead to degradation of the polymer carrier. It is also unclear whether it is possible to assess the CIC structure without implantation and the complexity of visualizing a non-contrast CIC sample in a gel for freezing during the subsequent production of a cryosection.

Among the analogs of our method, the development described in the works of Caroline D. Hoemann Michael D. Buschmann Marc D. McKee [11], Angros, Lee H [12], and Kang, Yujian James [13] deserves special attention. This group of methods describes in detail the process of preparing classical histological preparations. It contains a special adhesive polylysine layer on glass slides or an external limiter that fixes the histological section on the glass surface. The methods describe the process of preparation and histological analysis of CIC both before implantation in a model animal and after it as part of tissue blocks. These techniques demonstrate the possibility of specialized staining of the histological section for further confocal microscopy. Also, in the method of Kang, Yujian James [13], the fundamental possibility of performing CIC cryosection without specifying the technique is indicated. However, all of these methods include the process of creating paraffin blocks and reverse deparaffinization of the sample, which can lead to degradation of the CEC sample, in addition, there are no recommendations for the formation of a polylysine layer, the insufficient thickness of which can disrupt the fixation of the cut of the studied sample to the slide.

In Teixeira, S., Eblagon, K.M., Miranda, F., Pereira, M.F.R., Figueiredo, J.L. [3] showed that classical histological wiring, paraffinization and deparaffinization lead to the degradation of polylactide components in general and CIC based on it in particular. Also, the process of coloring in the presence of alcohol and xylene leads to degradation. These data make it possible to form one of the main shortcomings of the existing methods of sample preparation of the studied samples for histological examination - the degradation of the polymer carrier as part of the CMC design. However, the authors of this work do not offer solutions to eliminate the degradation of the scaffold or the finished CMC during the manufacture of a histological specimen.

The closest invention (prototype) to our method is the method described in the work of Prien, Samuel D., Blanton, John, Wood, Brian, Cassell, Allan J. [14]. It uses not standard histological wiring, but freezing samples at a temperature of -20 to -30 ° C, followed by cutting. This technique protects the samples from partial degradation and allows specific staining of cryosamples. However, the technique does not imply the possibility of studying CIC at the development stage and prior to implantation in an experimental animal, that is, outside the tissue block. There is also no indication of the possibility of preparing micropreparations for subsequent analysis on a confocal microscope, while insufficiently fast freezing of a CEC inhabited by eukaryotic cells at a temperature of -20 to -30°C is accompanied by the formation of ice microcrystals and leads to damage to the intracellular structures of eukaryotic cells, as well as makes their subsequent staining and confocal microscopy impossible. In addition, insufficient fixation to a glass slide during microtomy of a native or already populated scaffold can lead to damage (crumpling) of its structure.

It should be noted that the practical use of known methods is associated with the degradation of the polymer carrier and CMC based on polylactide during deparaffinization and subsequent staining, weak adhesion of the cryosection to the glass slide, slow freezing of the sample, which leads to inhomogeneous freezing and the lack of estimates of the possible degradation of the polymer scaffold during deparaffinization. and wiring for alcohols.

Thus, the common disadvantages of both the listed analogues and the prototype are: the use of alcohols and xylene for deparaffinization, which leads to partial degradation of the polymer carrier and impairs the analysis of CEC samples, both before implantation and as part of tissue blocks; the presence of a thin layer of polylysine on glass slides is the cause of weak adhesion and possible damage to the structure of the scaffold in case of its insufficient fixation; slow freezing of the sample at a temperature (-20-30°C) is accompanied by the formation of ice microcrystals and damage to the intracellular structures of eukaryotic cells, which does not allow confocal microscopy; the difficulty of visualizing a sample of a bright CIC during fixation in a cryo-gel for further production of a cryosection.

The objective of the invention is to develop a method for preparing a histological section of a CEC that allows preserving its structure, as well as a polylactide-based scaffold for further histological analysis of a cell-engineered construct and cell proliferation processes in it.

To solve the problem, the following experiments were carried out:

1. Increasing the adhesive ability of the glass slide due to the additional polylysine coating.

An additional adhesive layer of polylysine was applied to the surface of the slide and evenly distributed, after which the slides were placed in a thermostat at a temperature of +37°C for 24 hours. For one glass slide, 250 μl of polylysine at a concentration of 1 mg/ml was used. Thus, an additional adhesive (polylysine) layer was created on the surface of the slide for reliable fixation of the future cryosection and its subsequent coloring. In the absence of an additional layer, the CIC cryosection did not adhere reliably to the glass slide and the section peeled off during subsequent staining.

2. Creation and pre-staining of the CFC to enhance its contrast in the fixing gel for freezing.

At the second stage, cell-engineering constructs were made. A polymer frame made of polylactide was taken as a basis (Figure 1). The cell component was a culture of mesenchymal multipotent stromal bone marrow cells obtained from the bone marrow of a mature rat. The cells were obtained at the Center for Collective Use “Collection of Vertebrate Cell Cultures” of the Institute of Scientific Centers of the Russian Academy of Sciences [16]. The cell culture was dynamically combined with the polymer carrier using a device previously developed for this purpose [15]. KIK were cultivated under standard conditions for 21 days in Dulbecco's Modified Eagle Medium with the addition of a mixture of antibiotics penicillin-streptomycin (final concentration 50 μg/ml) and Fetal Bovine Serum serum (final concentration 10%). The preparation of histological sections for the analysis of cell proliferation inside the CEC was performed on the 14th and 21st days of observation. CIC samples at the indicated time to increase the contrast and better visualization of the scaffold were stained with an aqueous solution of 1% eosin for 20 minutes, which contributed to a more accurate positioning of the tested CIC samples in the gel for fixation (Figure 2). Without staining the cryosection of the CFC, it is not possible to visualize the CFC in the poured compound and to carry out the initial trimming without the risk of losing part of the sample.

3. Production of a CIC cryosection and its histological examination using light and confocal microscopy.

The CEC sample was fixed on an object stage and filled with TissueTek® cryomicroscopy compound (Leica Biosystems, Germany) until the sample was completely covered. After that, the CIC sample with the gel was placed in liquid nitrogen for shock cooling. 1 minute after complete cooling, the sample was placed in a cryomicrotome, where the sample was cut at a temperature of -20°C. The cut thickness was from 10 to 20 µm. The initial trimming was carried out to the base of the CIC stained with eosin, which contrasted against the background of a light background of the gel. Next, the area of interest was cut and the resulting sections were fixed on a glass slide preliminarily modified with polylysine. The resulting sections were stained with various dyes (hematoxylin and eosin, alcian blue) and covered with coverslips for subsequent microscopy. During the preparation of histological preparations, the use of alcohol and xylene was excluded. It was found that the resulting sections give an informative picture of the processes of proliferation of the MMSC cell culture inside the biodegradable carrier (Figure 5). It was also shown that the cell culture fills all the cavities of the porous carrier by the 14th day of observation. On the terms of 14 and 21 days, when stained with alcian blue, the presence of synthesized proteins of the extracellular matrix of hyaline cartilage was noted. During fluorescent and confocal microscopy of cryosections stained with DAPI, a clear visualization of the nuclear elements of the cells was obtained. Data were obtained on the three-dimensional distribution of the cell culture inside the CEC, as well as on the nature of the filling of the MMSC scaffold. There were no signs of degradation of cryosections during their production and staining (Figure 6.7). Without blast chilling, we were not able to achieve a stable CFC crisis.

4. SEM analysis of CIC and histological examination of CIC using the paraffin block technique were used as comparison methods.

Comparison methods were classical histological sample preparation and SEM (Figure 5). Data were obtained from standard histological waxing and dewaxing in xylene and ascending alcohol concentrations. It was found that alcohols and xylene lead to the hydrolysis of the scaffold from polylactic acid (Figure 3.4), which is consistent with data from the scientific literature.

SEM was carried out on a Jeol JSM6390LA hardware complex (Jeol, Japan) in cooperation with the FGBUN Botanical Institute named after N.N. V.L. Komarov. Tested CEC samples with dimensions not exceeding 8 x 8 x 8 mm were dehydrated and dried under vacuum. Next, a layer of palladium and gold with a thickness of 5 nm was sputtered onto the preparation, and then the CIC surface was analyzed (Figure 5). The result made it possible to obtain the numerical values of the studied samples, the surface structure, and to evaluate the perifocal region of interest.

Thus, in the study a new method of CEC sample preparation based on polylactic acid and eukaryotic cell culture was identified for histological analysis of CEC and cell proliferation processes in it, as a result of which high-quality histological cryopreparations were obtained with preservation of the scaffold structure, analyzed on a fluorescent and confocal microscope without signs of degradation and the use of alcohols and xylene, as well as dewaxing.

The technical result is to ensure the manufacture of high-quality histological CEC preparations, namely, the preservation of the structure of the scaffold and the integrity of intracellular structures, which makes it possible to evaluate the interaction of the polymer carrier and the cell culture, as well as the proliferation of the latter.

The technical result is achieved due to the fact that for the study of the sample, glasses are preliminarily prepared with an additional polylysine layer of 250 μl of polylysine, evenly applied on top of each glass slide at a concentration of 1 mg/ml, then the glasses are dried for 24 hours at a temperature of 37°C; a sample of a cell-engineering construct is preliminarily stained with a 1% solution of eosin, after which it is shock-frozen in liquid nitrogen at a temperature of minus 196 °C, fixed in a cryomicrotome using cryogel, then a cryomicroscopic cutting of the area of the cell-engineered construct is carried out with its fixation on the prepared adhesive glass slide.

The application of an additional adhesive layer on the surface of a glass slide, contrast tinting of CIC with eosin, and shock (emergency) freezing in liquid nitrogen avoid the use of alcohols, xylene, and deparaffinization, the use of which leads to the degradation of the polymer carrier and does not allow one to effectively assess the ongoing processes.

Simultaneous use of shock freezing of a CIC sample in liquid nitrogen and a preliminarily applied additional adhesive polylysine layer on a glass slide eliminates the degradation of the polylactide carrier sample from the cell culture, and also allows for specific histochemical staining and analysis of images on a confocal microscope both before and after CIC implantation in organism of experimental animals (rats).

The figures show:

Figure 1. Photograph of a polylactic acid scaffold blank for creating a CFC.

Figure 2. Photo of CIC after counterstaining with eosin. Dynamic colonization of biodegradable polylactide carrier by cell culture.

Figure 3. Histological preparation of CIC based on polylactide and cell culture. The figure shows the CIC after standard histological wiring, including deparaffinization, xylene and alcohol treatments. Sites of scaffold degradation from polylactic acid are identified.

Figure 4. Histological preparation of CIC based on polylactide and cell culture. The figure shows the CIC after standard histological wiring, including deparaffinization, xylene and alcohol treatments. Sites of scaffold degradation from polylactic acid are identified.

Figure 5. SEM image of CIC immediately after cell colonization. The figure shows the surface of the CIC. View from above. The control stroke is indicated in the figure.

Figure 6. Histological section of CIC after cultivation. The figure shows histological images of cryosections stained with histological dyes. A, B - 7 days after settlement. Figure B corresponds to the selected area of Figure A. Figure C - after 14 days after colonization and cultivation. Alcian blue color. The control stroke is indicated in the figure.

Figure 7. Cryocut KIK. The figure shows histological images of cryosections obtained on a fluorescent (A, B) and confocal (C) microscope. The control stroke is indicated in the figure.

Figure 8. Visualization of the location of cells inside a biodegradable scaffold in three-dimensional space obtained by making cryosections and subsequent confocal microscopy.

Figure 9. The location of the cells of the boundary layer during the dynamic settlement of the scaffold. Cryosection and confocal microscopy. The control stroke is indicated in the figure.

The method is carried out as follows: a cell culture of mesenchymal multipotent stromal bone marrow cells is isolated from a sexually mature rat. The culture is passaged up to passage 3 and checked on a flow cytometer for the presence of surface markers of CD90 expression and the absence of CD45 markers.

Then a cell-engineered structure is created. First, a polymer carrier based on polylactic acid (PLA) is prepared, then it is sterilized, and the polylactide form is pre-treated for future combination with the cell culture in a dynamic way in the developed bioreactor. After this procedure, the cylindrical molds are sterilized and placed in a bioreactor for dynamic colonization of the cell culture. A cell culture with a volume of 5 ml and a concentration of 10 ⁶ viable cells per 1 ml is pumped twice through a biodegradable polylactide scaffold. Thus, the scaffold is populated with cells to the entire depth and over the entire surface.

Next, the KIK cultivated for 21 days with the addition of the required amount of nutrient medium for cell culture, serum and antibiotic.

Before preparing the cryosection, glass slides are prepared to make their surface more adhesive. To do this, the glass surface is additionally treated with polylysine (on top of the regular polylysine layer, which has already been applied by the manufacturer of these glasses). Glasses are incubated in a thermostat at +37°C for an hour, after which they are ready for use.

The samples of the obtained CECs based on polylactide have a light color to contrast them against the background of the flooding and fixing TissueTek® gel (Leica Biosystem, Germany), they are preliminarily stained with eosin. The stained CECs are poured with a fixing gel on a cryosection platform, then the structure is placed in liquid nitrogen for shock freezing at a temperature of -196°C.

Trimming of the frozen gel takes place at -20°C to the colored area of the CIC. Cryosections are made with a thickness of 10-20 μm and fixed on the adhesive surface of a glass slide with polylysine, which is pre-applied at a concentration of 1 mg per ml in an amount of 250 μl per glass and distributed evenly. After applying a layer of polylysine, the glass is dried at a temperature of +37°C for 24 hours. The obtained CICs are used to assess the proliferation of a cell culture inside a biodegradable polymer scaffold by filling and distributing cells inside the polymer. Based on the analysis of cell culture-free zones in the polymer at different periods of observation, the degree and direction of proliferation and filling of free space inside the porous polylactide carrier, as well as the dynamics of filling free spaces with cell culture, are assessed.

Example.

For the experiment, a cell culture was taken from the collection of cell cultures of vertebrates of the Center for Collective Use of the Institute of Cytology of the Russian Academy of Sciences and a cell-engineering construct was created from a polymer based on polylactic acid. Cryosections were obtained from CIC samples for further analysis. Based on the CIC cryosections obtained using layer-by-layer confocal microscopy with staining for cell nuclei (DAPI), high-quality histological cryosections were obtained in the experiments, and the integrity of the polymer scaffold, the viability and location of cells in the central and border regions of the polymer scaffold, as well as the uniformity, direction and the dynamics of their proliferation when using different periods of observation. Thus, it was noted that the cell culture proliferates and fills the free areas of the polymer, and the degree of filling increases with the observation period. Using 3D visualization, the integrity of cellular structures, cell proliferation in different layers and areas in one analyzed sample was assessed. It was found that the maximum concentration of cells after dynamic colonization of the polymer scaffold by the cell culture is formed in the central region and in the boundary layer. The location of cells within a three-dimensional biodegradable carrier is shown using the technique of making cryosections and confocal microscopy (Figure 8, Figure 9). Using confocal microscopy and specific DAPI staining, it was found that in a cross section of a cell-engineered construct, the distribution of cells is the same from layer to layer. Thus, it can be concluded that the result has been achieved.

Bibliography.

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16. The cell culture was obtained at the Center for Collective Use in the Collection of Vertebrate Cell Cultures at the Institute of Cytology, Russian Academy of Sciences.

Claims (1)

A method for sample preparation of a cell-engineering construct based on polylactic acid and a culture of eukaryotic cells for histological analysis, including a sequence of actions for freezing the test sample, performing histological sections on a cryomicrotome, followed by making micropreparations and their histochemical staining, characterized in that slides are preliminarily prepared on each of which is uniformly applied on top of a polylysine layer of 250 μl of polylysine at a concentration of 1 mg / ml, and dried for 24 hours at a temperature of 37 ° C; a sample of a cell-engineering construct is preliminarily stained with a 1% solution  eosin, after which it is shock-frozen in liquid nitrogen at a temperature of minus 196 °C, then it is fixed in a cryomicrotome using cryogel, then a cryomicroscopic excision of the area of the cell-engineered construct is performed, after which it is fixed it on a pre-prepared adhesive slide.

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