<meta charset="utf8"></head><body>高效的 3D 皮肤模型，用于灵活的研究应用

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>24-well plate for adherent cell culture</td>
			<td>Biologix Europe GmbH</td>
			<td>07-6024</td>
			<td>-</td>
		</tr>
		<tr>
			<td>35%&#8211;38% HCL</td>
			<td>Chempur</td>
			<td>115752837</td>
			<td>-</td>
		</tr>
		<tr>
			<td>60 mm cell culture Petri dish&#160;</td>
			<td>Nest</td>
			<td>705001</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Avidin&#8722;Sulforhodamine 101</td>
			<td>Sigma Aldrich</td>
			<td>A2348-5MG</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Bright-field inverted microscope</td>
			<td>Olympus</td>
			<td>CKX41</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Calcium chloride</td>
			<td>Avantor</td>
			<td>874870116</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Cell culture flask T75 for adherent cells</td>
			<td>Genoplast</td>
			<td>G77080033</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Centrifuge tube 15 mL</td>
			<td>GoogLab Scientific</td>
			<td>G66010522</td>
			<td>-</td>
		</tr>
		<tr>
			<td>CO2 Incubator</td>
			<td>Heal Force</td>
			<td>Galaxy 170R</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Col1A2 antibody produced in rabbit</td>
			<td>Novus</td>
			<td>NBP2-92790</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Corning(R) Transwell(R) Polycarbonate</td>
			<td>Corning</td>
			<td>CLS3422-48EA</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Cytokeratin 14 antibody produced in mouse</td>
			<td>Novus</td>
			<td>NBP1-79069</td>
			<td>-</td>
		</tr>
		<tr>
			<td>DPX Mountant for histology</td>
			<td>Sigma Aldrich</td>
			<td>06522-100ML</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle Medium (DMEM)</td>
			<td>VWR Chemicals</td>
			<td>L0102-500</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Eosine Y</td>
			<td>Kolchem</td>
			<td>-</td>
			<td>0.5 % aquatic solution</td>
		</tr>
		<tr>
			<td>Eppendorf tube 1.5 mL</td>
			<td>Sarstedt</td>
			<td>72.690.001</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Eppendorf tube 2 mL</td>
			<td>Sarstedt</td>
			<td>72.691</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Ethyl alcohol absolute 99.8%</td>
			<td>Avantor</td>
			<td>396480111</td>
			<td>diluted in ultrapure water to the needed concentrations&#160;</td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Gibco</td>
			<td>10270106</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Fluorescent inverted microscope&#160;</td>
			<td>Olympus</td>
			<td>IX71</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Goat anti-mouse secondary antibody conjugated with FITC</td>
			<td>Sigma Aldrich</td>
			<td>F0257-1mL</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-rabbit secondary antibody conjugated with FITC</td>
			<td>Novus</td>
			<td>NB7159</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Harris Hematoxylin</td>
			<td>Kolchem</td>
			<td>-</td>
			<td>1 mg/mL in 95% ethanol</td>
		</tr>
		<tr>
			<td>Hoechst 33342</td>
			<td>ThermoFisher</td>
			<td>H3570</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Laminar chamber</td>
			<td>Heal Force</td>
			<td>HFSafe-1200</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Melan-A antibody produced in mouse</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-20032</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Microtome</td>
			<td>Microm</td>
			<td>HM355S</td>
			<td>-</td>
		</tr>
		<tr>
			<td>NaOH&#160;</td>
			<td>Avantor</td>
			<td>810981997</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Paraffin pastilles</td>
			<td>Sigma Aldrich</td>
			<td>1.07164</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sigma Aldrich</td>
			<td>1581227</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin solution</td>
			<td>Sigma Aldrich</td>
			<td>P4333</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Pipette tip, 1000 &#181;L</td>
			<td>Sarstedt</td>
			<td>70.305</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Pipette tip, 20 &#181;L</td>
			<td>Sarstedt</td>
			<td>70.3021</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Pipette tip, 200 &#181;L</td>
			<td>Sarstedt</td>
			<td>70.303</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Pluronic F-127</td>
			<td>BASF</td>
			<td>50401036</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Serological pipette 10 mL</td>
			<td>GoogLab Scientific</td>
			<td>G33270011</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Serological pipette 25 mL</td>
			<td>GoogLab Scientific</td>
			<td>G33280011</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Serological pipette 5 mL</td>
			<td>GoogLab Scientific</td>
			<td>G33260011</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Sodium bicarbonate</td>
			<td>Sigma Aldrich</td>
			<td>S5761</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Sodium bicarbonate</td>
			<td>Chempur</td>
			<td>118105307</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA 0.25% solution, phenol red</td>
			<td>Sigma Aldrich</td>
			<td>25200072</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Type 1 collagen</td>
			<td>IBIDI</td>
			<td>50201</td>
			<td>-</td>
		</tr>
		<tr>
			<td>U-bottom 96-well plate</td>
			<td>Sarstedt</td>
			<td>83.3925500</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Xylene</td>
			<td>Sigma Aldrich</td>
			<td>534056</td>
			<td>-</td>
		</tr>
	</tbody>
</table>

building up skin models for numerous applications - from two-dimensional (2d) monoculture to three-dimensional (3d) multiculture

The skin is a continuous structure with multicell interactions revealing the proper functioning and homeostasis of this complex organ. It is built from morphologically different layers: the inner layer - dermis, and the outer layer - the epidermis. On top of the epidermis, we additionally distinguish the stratum corneum&#160;(consisting of flattened dead cells - corneocytes), which provides the greatest protection against the external environment. Some of the most important passive and active functions of the skin are body protection against external factors, participation in the immunological processes, secretion, resorption, thermoregulation, and sensing1,2,3. Because it is considered one of the largest organs in the body, it is impossible to avoid contact with various pathogens, allergens, chemicals, as well as ultraviolet (UV) radiation. Thus, it is structured with many types of cells with specific functions. The main types of cells present in the epidermis are keratinocytes (almost 90% of all cells, with structural and immunological functions in the deeper parts of the epidermis, but which later undergo the keratinization process to turn to corneocytes in the top layer of the epidermis), melanocytes (only 3%-7% of the epidermal cell population, which produce the UV protective pigment melanin) and the Langerhans cells (from the immune system). In the case of the dermis, the main cells are fibroblasts (producing growth factors and proteins), dendritic cells, and mast cells (both cell types of the immune system)4,5,6. Moreover, the skin is equipped with several extracellular proteins (such as collagen type I and IV, fibronectin, and laminin; Figure 1) and protein fibers (collagen and elastin), which ensure the specific structure of the skin but also encourage cell binding, cell adhesion, and other interactions7.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65773/65773fig01.jpg" /> 
Figure 1: Schematic showing the skin structure. The skin structure marked four basic cell types occurring in its individual layers and distinguished proteins of the extracellular matrix. This figure was created with MS PowerPoint. <a href="https://www.jove.com/files/ftp_upload/65773/65773fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The safety of cosmetics and pharmaceutical products is a very important issue, and protecting the health of consumers and patients is a priority8. Until recently, it was supposed to be guaranteed by numerous tests, including studies conducted on animals. Unfortunately, these often required the use of drastic methods, causing pain and suffering in animals used for research purposes (frequently mice, rats, and pigs). In 1959, the Principles of the Humane Experimental Technique (the 3Rs principle) were introduced: (1 - Replacement) replacing animals in research with in vitro, in sillico, or ex vivo models, (2 - Reduction) reducing the number of animals used for research, and (3 - Refinement) improving the well-being of the animals which are still needed for research and at the same time improving the developed alternative methods9. Furthermore, in the European Union (EU), cosmetic testing on animals is regulated by law. From September 11, 2004 the ban on animal-tested cosmetic products came into force. On March 11, 2009, the EU banned animal testing of cosmetic ingredients. The sale of cosmetic products made of newly animal-tested ingredients was not allowed; however, testing the products on animals for complex human health issues such as repeat dose toxicity, reproductive toxicity, and toxicokinetics was still acceptable. Starting from March 11, 2013, in the EU, it is illegal to sell cosmetics where the finished product or its ingredients have been tested on animals10. Therefore, currently, in cosmetology, research is carried out at three levels: in vitro (cells), ex vivo (real tissues), and in vivo (volunteers)11. In the case of pharmaceuticals, the need for animal testing remains; however, it is significantly reduced and strictly controlled12.
As alternative methods to animal testing and for the initial assessment of the effectiveness of a novel active ingredient, the in vitro skin cell cultures are used. Isolation of different types of skin cells and their cultivation in sterile laboratory conditions allows one to assess the safety and toxicity of active substances. Skin cell lines are also widely recognized models for research as the cells are sold by certified companies and the results can be comparable in different laboratories. These tests are usually performed on simple 2D models of the human skin cell monocultures. Some of the more advanced models are their co-cultures (such as keratinocytes with fibroblasts and keratinocytes with melanocytes), as well as the three-dimensional models, including scaffold-free cultures (spheres) and scaffold-based skin equivalents of the epidermis, dermis or even the full-thickness substitutes of the skin13. It is worth mentioning that apart from the last type (skin equivalents), the rest are not commercially available, and if needed, a scientist must prepare them on his/her own.
Even though plenty of these models have been maintained and are routinely sold nowadays (Table 1), additional models are constantly needed to validate most of the results. Thus, the newly engineered models ought to recreate better the real interactions taking place in the human body. When a mixture of cells of different types is used to form such models, the reproduction of the multicellular aspect of tissue in vivo can be achieved. As a result, an organotypic culture is developed (Figure 2).
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td colspan="2">Name</td>
			<td colspan="4">Description</td>
		</tr>
		<tr>
			<td rowspan="6">Normal skin</td>
			<td colspan="2">EpiSkin</td>
			<td colspan="4">Reconstructed Human Epidermis - Keratinocytes on a collagen membrane</td>
		</tr>
		<tr>
			<td colspan="2">SkinEthic RHE</td>
			<td colspan="4">Reconstructed Human Epidermis - Keratinocytes on a polycarbonate membrane</td>
		</tr>
		<tr>
			<td colspan="2">SkinEthic RHE-LC</td>
			<td colspan="4">Human Epidermal Model Langerhans Cells - Keratinocytes and Langerhans cells on a polycarbonate membrane</td>
		</tr>
		<tr>
			<td colspan="2">SkinEthic RHPE</td>
			<td colspan="4">Reconstructed Human Pigmented Epidermis - Keratinocytes and Melanocytes on a polycarbonate membrane</td>
		</tr>
		<tr>
			<td colspan="2">T-Skin</td>
			<td colspan="4">Reconstructed Human Full Thickness Skin Model - Keratinocytes on a layer of Fibroblasts, which were grown on a polycarbonate membrane</td>
		</tr>
		<tr>
			<td colspan="2">Phenion FT Skin model</td>
			<td colspan="4">Keratinocytes and Fibroblasts in hydrogel</td>
		</tr>
		<tr>
			<td rowspan="2">Skin with a disease</td>
			<td colspan="2">Melanoma FT Skin Model</td>
			<td colspan="4">Normal human-derived Keratinocytes and Fibroblasts with human Malignant Melanoma cell line A375</td>
		</tr>
		<tr>
			<td colspan="2">Psoriasis Tissue Model</td>
			<td colspan="4">Normal human Keratinocytes and Fibroblasts</td>
		</tr>
	</tbody>
</table>
Table 1: The most popular commercial skin equivalents for various studies.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65773/65773fig02.jpg" /> 
Figure 2: Complexity of different in vitro models. The relation between the complexity of different in vitro models to recreate an organism and the real interactions occurring directly in the human body. The figure has been modified from the set &#34;Microbiology and cell culture&#34; from Servier Medical Art by Servier (https://smart.servier.com/).&#160;<a href="https://www.jove.com/files/ftp_upload/65773/65773fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
One of the most important limitations of the commercial equivalents is the availability of only very general research models with a few types of cells (typically 1-2, seldom 3). Yet, there are many more cells present in the skin, and their interaction with each other can ensure better or worse tolerance of various ingredients14. The lack of some immune components can decrease its value in several kinds of research, including immunotherapy. This is a serious issue as melanoma is a life-threatening skin cancer due to the early onset of metastasis and frequent resistance to the applied treatment15. To improve the artificial skin model, researchers try to establish a co-culture of immune cells with cell lines and organoids16&#160;and this is considered a big improvement of the studied models. For example, mast cells take part in many physiological (wound healing, tissue remodeling) and pathological (inflammation, angiogenesis, and tumor progression) processes in the skin17. Thus, their occurrence in the model can significantly change the model&#39;s response to the studied compound. Finally, a lot of skin-related information is still missing, which can only be discovered by performing basic research. This is why creating and refining different artificial skin models (Table 2) is such an important endeavor. This article presents several procedures to create advanced skin models, including spheres and skin equivalents.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>In vitro skin model</td>
			<td>Attempt to recreate interactions occurring in the tissue</td>
			<td>Examples of the used cells</td>
		</tr>
		<tr>
			<td rowspan="12">2D or 3D cell culture</td>
			<td rowspan="3">Epidermis</td>
			<td>Keratinocytes</td>
		</tr>
		<tr>
			<td>Melanocytes</td>
		</tr>
		<tr>
			<td>Keratinocytes + Melanocytes</td>
		</tr>
		<tr>
			<td rowspan="3">Dermis</td>
			<td>Fibroblasts</td>
		</tr>
		<tr>
			<td>Mast cells</td>
		</tr>
		<tr>
			<td>Fibroblasts + Mast cells</td>
		</tr>
		<tr>
			<td rowspan="6">Skin</td>
			<td>Keratinocytes + Fibroblasts</td>
		</tr>
		<tr>
			<td>Keratinocytes + Mast cells</td>
		</tr>
		<tr>
			<td>Melanocytes + Fibroblasts</td>
		</tr>
		<tr>
			<td>Melanocytes + Mast cells</td>
		</tr>
		<tr>
			<td>Keratinocytes + Fibroblasts + Melanocytes</td>
		</tr>
		<tr>
			<td>Keratinocytes + Fibroblasts + Mast cells</td>
		</tr>
	</tbody>
</table>
Table 2: Examples of cell type mixture to recreate skin tissue in 2D and 3D culture.

<meta charset="utf8"></head><body>作者聚焦：使用经济高效的 3D 细胞模型增强皮肤模型的多样性

为众多应用构建皮肤模型 - 从二维 （2D） 单一培养到三维 （3D） 多培养

Our challenge is to test ingredients'cytotoxicity prior to cosmetic or medical use. The commercial 3-D skin models limited to around three cell types don't reflect real skin complexity. And skin has diverse cell types with essential functions, posing a challenge in accurately assessing ingredient impact.Our findings indicate that the limiting cell adhesion method produced more well-shaped spheres compared to the commonly-used methods. Additionally, it demands less manipulation, ensuring greater sphere stability. In our skin equivalent, we use up to four types of skin cells, unlike the commercial models that typically incorporate a maximum of three cell types.Common to these cell models lack accuracy in representing real interaction. While commercial 3-D skin equivalents offer better tissue representation, their cost limits widespread use. Consequently, our self-established models allow for initial cost-effective experiments on a more realistic platform.Our protocols outline affordable, straightforward ways to create customized 3-D skin models, encompassing spheres and equivalents. In contrast to commercial options, our models offer adjustable appearance and composition tailored to the study's requirements. The method is accessible, not demanding specialized skills or equipment, making them replicable even by novice researchers.Our aim is to highlight the simplicity of creating various 3-D cellular models, while emphasizing the value of certified commercial models in research. We stress the significance of using realistic models, such as spheres and equivalents. Showcasing how the cells make models, can aid in pre-selecting the testing conditions before employing certified models.

Watch this Scientific Journal Video about Building Up Skin Models for Numerous Applications - from Two-Dimensional 2D Monoculture to Three-Dimensional 3D Multiculture at JoVE.com

Due to the complex structure and important functions of the skin, it is an interesting research model for the cosmetic, pharmaceutical, and medical ...

building-up-skin-models-for-numerous-applications-from-two

Research

JoVE Journal

Bioengineering

Building Up Skin Models for Numerous Applications - from Two-Dimensional (2D) Monoculture to Three-Dimensional (3D) Multiculture

1.4K 次观看.  Warsaw University of Technology. 我们面临的挑战是在化妆品或医疗用途之前测试成分的细胞毒性。仅限于大约三种细胞类型的商业 3-D 皮肤模型并不能反映真实的皮肤复杂性。皮肤具有多种具有基本功能的细胞类型，因此对准确评估成分影响构成了挑战。我们的研究结果表明，与常用方法相比，限制细胞粘附方法产生了更多形状良好的球体。此外，它需要更少的操作，确保更?...

视频: 作者聚焦：使用经济高效的 3D 细胞模型增强皮肤模型的多样性

Due to the complex structure and important functions of the skin, it is an interesting research model for the cosmetic, pharmaceutical, and medical industries. In the European Union, there has been a total ban on testing cosmetic products and their ingredients on animals. In the case of medicine and pharmaceuticals, this possibility is also constantly limited. In accordance with the 3Rs principle, it is becoming more and more common to test individual compounds as well as entire formulations on artificially created models. The cheapest and most widely used are the 2D models, which consist of a cell monolayer but do not reflect the real interactions between the cells in the tissue. Although the commercially available 3D models provide a better representation of the tissue, they are not used on a large scale. This is because they are expensive, the waiting time is quite long, and the available models are frequently limited to only those typically used. 
In order to move the conducted research to a higher level, we have optimized the procedures of various 3D skin model preparations. The described procedures are cheap and simple to prepare as they can be applied in numerous laboratories and by researchers with different experiences in cell culture.

Here, we present inexpensive and simple procedures to introduce various 3D skin models for routine research in a cell culture laboratory. Researchers can create models tailored to their needs without relying on commercially available models.

科研

生物工程

Preparing a Cell Suspension from the Adherent Skin Cell Cultures

To begin, remove the respective medium from the culture flasks containing the keratinocytes, fibroblasts, and melanocytes. Gently wash the cells with five to 10 milliliters of PBS. Now add 0.5 to two milliliters of Trypsin-EDTA solution to the flask, depending on its size.Incubate the flask at 37 degrees Celsius. Use a microscope to control the detachment of cells from the surface. Suspend the detached cells in double the volume of Trypsin Neutralizer to deactivate Trypsin.Then transfer the suspension into a 15-milliliter tube. Now pipette about 20 microliters of the skin cell suspension into a 1.5-milliliter tube. With the help of a hemocytometer, count the cells Next, centrifuge the tube at 300 g for five minutes at room temperature.Then pipette out most of the supernatant and resuspend the cell pellet in the small amount of remaining liquid. Add an equal volume of fresh medium to the resuspended cells. In a separate 15-milliliter tube, prepare cell suspension.The primary cells of keratinocytes, melanocytes, fibroblasts, and mast cells presented their typical morphology when grown in their respective media.

从贴壁皮肤细胞培养物制备细胞悬液

Preparation of Skin Cell Spheres for 3D Multiculture

To begin the hanging drop method, pipette 20 microliters of a pre-prepared skin cell suspension onto the lid of a Petri dish. Now, cover the lid with the bottom half of the Petri dish and gently flip it to create the hanging drops. Add sterile water to the Petri dish to prevent evaporation of the full-grown medium from droplets.Incubate the droplets for 48 to 72 hours at 37 degrees Celsius. Next, fill the wells of a new plate with the full growth medium. Using sterile cut pipette tips, transfer the cell spheres to the wells of the multi-well plate.Incubate the transferred spheres for one day in the multi-well plate at 37 degrees Celsius before experimentation. For the limiting cell adhesion method, first add 100 microliters of 1%surfactant solution in PBS into each well of a U-bottom plate. Next, incubate the plate with the solution at 37 degrees Celsius for 24 hours.Now, prepare the cell suspension in the desired cell density. Pipette out the surfactant solution from the wells, then seed 50 microliters of the cell suspension into each well. Finally, incubate the plate at 37 degrees Celsius for 24 hours to reach cell aggregates.Spheres consisting of 10, 000 cells were easier to manipulate, as they were visible in the wells. The bigger spheres showed decreased stability. Different cell types formed spheres of different colors.The perfectly rounded spheres were prone to losing their shape during the transfer in the hanging drop method. Accurate handling was required to reduce the deformation resulting in intact cell aggregates. Inaccuracy and inexperience resulted in sphere damage.

用于 3D 多培养的皮肤细胞球的制备

Preparation of Full-Thickness Skin Equivalents for 3D Multiculture

To begin, mix water, 10 times concentrated PBS, and one molar sodium hydroxide in a tube. Next, pipette 500 microliters of the appropriately dense dermal cell solutions to a two milliliter tube. Centrifuge the solution at 300g for three minutes at room temperature.Once centrifugation is complete, pipette out the supernatant and gently resuspend the cells in a mixture of water, PBS, and sodium hydroxide. Now, add 200 microliters of the collagen solution to the mixture. Pipette the suspension to mix it well.Next, pipette 500 microliters of the prepared mixture into an insert in a 24 well plate. For a model without the stratum corneum, pipette 500 microliters of the mixture to a well without an insert. After incubating the plate for 10 minutes at room temperature, transfer it to an incubator for 30 minutes.Pipette 500 microliters of PBS buffered into each well to rinse the hydrogel surface. Next, mix 200 microliters of the keratinocyte suspension with an equal volume of melanocyte suspension in the supplemented DMEM medium. Gently pipette 500 microliters of the total cell suspension into each well.Incubate the plates at 37 degrees Celsius for two to five days. Replace the medium every 48 hours and use an optical microscope to monitor the cell growth. An equivalent 3D model with distinguishable dermis and epidermis was created, which could be monitored in real time.Keratinocytes in different stages of cell growth were observed, which are indicators of a living equivalent. Mast cells with recognizable granules were also seen.