Name: idg-sw3 cell culture in a three-dimensional extracellular matrix
Uploaded: 2023-11-13T14:10:00
Description: Watch this Scientific Journal Video about IDG-SW3 Cell Culture in a Three-Dimensional Extracellular Matrix at JoVE.com

हम आईडीजी-एसडब्ल्यू 3 सेल लाइन उपहार में देने के लिए डॉ लिंडा एफ बोनवाल्ड को धन्यवाद देते हैं। इस काम को चीन के राष्ट्रीय प्राकृतिक विज्ञान फाउंडेशन (82070902, 82100935 और 81700778) और शंघाई "विज्ञान और प्रौद्योगिकी नवाचार" नौकायन परियोजना (21वाईएफ 1442000) द्वारा समर्थित किया गया था।

यह प्रोटोकॉल 24-वेल प्लेटों के चार कुओं में कोशिकाओं के संवर्धन के लिए उपयुक्त है। यदि कई नमूने या प्लेटें तैयार कर रहे हैं, तो अभिकर्मकों की मात्रा तदनुसार बढ़ाई जानी चाहिए।
1. कोलेजन I मिश्र...

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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomineralization+in+humans:+making+the+hard+choices+in+life.">Biomineralization in humans: making the hard choices in life.</a> Annual Review of Genetics. 43, 119-142 (2009).</li><li>Bosman, F. T., Stamenkovic, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+structure+and+composition+of+the+extracellular+matrix.">Functional structure and composition of the extracellular matrix.</a> Journal of Pathology. 200 (4), 423-428 (2003).</li><li>Papadopoulos, N. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+improved+fluorescence+assay+for+the+determination+of+lymphocyte-mediated+cytotoxicity+using+flow+cytometry.">An improved fluorescence assay for the determination of lymphocyte-mediated cytotoxicity using flow cytometry.</a> Journal of Immunological Methods. 177 (1-2), 101-111 (1994).</li><li>Staines, K. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypomorphic+conditional+deletion+of+E11/Podoplanin+reveals+a+role+in+osteocyte+dendrite+elongation.">Hypomorphic conditional deletion of E11/Podoplanin reveals a role in osteocyte dendrite elongation.</a> Journal of Cellular Physiology. 232 (11), 3006-3019 (2017).</li><li>Feng, J. Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loss+of+DMP1+causes+rickets+and+osteomalacia+and+identifies+a+role+for+osteocytes+in+mineral+metabolism.">Loss of DMP1 causes rickets and osteomalacia and identifies a role for osteocytes in mineral metabolism.</a> Nature Genetics. 38 (11), 1310-1315 (2006).</li><li>Winkler, D. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Osteocyte+control+of+bone+formation+via+sclerostin,+a+novel+BMP+antagonist.">Osteocyte control of bone formation via sclerostin, a novel BMP antagonist.</a> EMBO Journal. 22 (23), 6267-6276 (2003).</li><li>Riminucci, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FGF-23+in+fibrous+dysplasia+of+bone+and+its+relationship+to+renal+phosphate+wasting.">FGF-23 in fibrous dysplasia of bone and its relationship to renal phosphate wasting.</a> Journal of Clinical Investigation. 112 (5), 683-692 (2003).</li><li>Dallas, S. L., Bonewald, L. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamics+of+the+transition+from+osteoblast+to+osteocyte.">Dynamics of the transition from osteoblast to osteocyte.</a> Annals of the New York Academy of Sciences. 1192, 437-443 (2010).</li><li>Robin, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Involvement+of+3D+osteoblast+migration+and+bone+apatite+during+in+vitro+early+osteocytogenesis.">Involvement of 3D osteoblast migration and bone apatite during in vitro early osteocytogenesis.</a> Bone. 88, 146-156 (2016).</li><li>Chen, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+mineralization+capacity+of+IDG-SW3+cells+in+3D+collagen+hydrogel+for+bone+healing+in+estrogen-deficient+mice.">High mineralization capacity of IDG-SW3 cells in 3D collagen hydrogel for bone healing in estrogen-deficient mice.</a> Frontiers in Bioengineering and Biotechnology. 8, 864 (2020).</li></ol>

इस प्रोटोकॉल में एक महत्वपूर्ण बिंदु यह है कि सहज जमावट को रोकने के लिए चरण 1 और चरण 2 को बर्फ पर किया जाना चाहिए। इस विधि में, कोलेजन I की अंतिम एकाग्रता 1.2 मिलीग्राम / इस प्रकार, विभिन्न निर्माताओं से विभिन?...

ओस्टियोसाइट्स ओस्टियोब्लास्ट 1,2 से प्राप्त टर्मिनल विभेदित कोशिकाएं हैं। एक बार ओस्टियोब्लास्ट को ओस्टियोइड द्वारा दफन कर दिया जाता है, तो यह ओस्टियोसाइटोजेनेसिस से गुजरता है और प्रीओस्टियोसाइट्स बनाने के लिए पीडीपीएन जीन को व्यक्त करता है, ओस्टियोइड को खनिज करने के लिए डीएमपी 1 जीन, और हड्डीके ऊतकों में परिपक्व ऑस्टियोसाइट के रूप में कार्य करने के लिए सोस्ट और एफजीएफ 23 जीन। यहां, ओस्टियोसाइटोजेनेसिस प्रक्रिया में डेंड्राइट एक्सटेंशन और मार्कर जीन अभिव्यक्ति की पहचान करने के लिए एक 3 डी संवर्धन प्रणाली पेश की गई है।
आईडीजी-एसडब्ल्यू 3 कोशिकाएं ट्रांसजेनिक चूहों से प्राप्त एक अमर प्राथमिक सेल लाइन हैं औरविभिन्न मीडिया 4 में सुसंस्कृत होने पर ओस्टियोसाइट भेदभाव के लिए ओस्टियोब्लास्ट का विस्तार या प्रतिकृति कर सकती हैं। एमएलओ-ए 5, एमएलओ-वाई 4, और अन्य सेल लाइनों की तुलना में, कार्यात्मक प्रोटीन की अभिव्यक्ति प्रोफ़ाइल, कैल्शियम नमक जमाव करने की क्षमता, और आईडीजी-एसडब्ल्यू 3 कोशिकाओं में विभिन्न हार्मोनों की प्रतिक्रियाएं हड्डीके ऊतकों में प्राथमिक ओस्टियोसाइट्स के समान होने की अधिक संभावना है।
2 डी सिस्टम की तुलना में, 3 डी संवर्धन प्रणाली विवो सेलुलर विकास वातावरण की नकल करने में अधिक सक्षम हैं, जिसमें पोषक तत्व ढाल, कम यांत्रिक कठोरता और आसपास की यांत्रिक सीमा शामिल है (तालिका 1)। 3 डी प्रणाली में ओस्टियोब्लास्टिक कोशिकाओं की खेती के लिए पिछले तरीकों में से अधिकांश ने कोलेजन I को फॉर्मूलेशन 4,5,6 में अद्वितीय घटक के रूप में उपयोग किया, क्योंकि कोलेजन I फाइबर कैल्शियम और फास्फोरस जमाव की साइट के रूप में काम करते हैं। हालांकि, ओस्टियोइड में एक अनिवार्य घटक, बाह्य मैट्रिक्स, में सेलुलर कारकों का एक बड़ा समूह होता है जो सेलुलर विकास, आसंजन और माइग्रेशन 7,8 को बढ़ावा देता है और इमेजिंग अवलोकन के लिए पारदर्शी और सुविधाजनक है। इस प्रकार, यह प्रोटोकॉल ओस्टियोसाइटोजेनेसिस अध्ययन के लिए एक माध्यमिक घटक के रूप में मैट्रिगेल (इसके बाद बेसमेंट झिल्ली मैट्रिक्स के रूप में संदर्भित) का उपयोग करता है।

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.25% Trypsin-ethylenediaminetetraacetic acid</td>
			<td>Hyclone</td>
			<td>SH30042.01</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL tubes</td>
			<td>Corning, NY, USA</td>
			<td>430791</td>
			<td></td>
		</tr>
		<tr>
			<td>7.5% (w/v) Sodium bicarbonate</td>
			<td>Sigma-Aldrich, MO, USA</td>
			<td>S8761</td>
			<td></td>
		</tr>
		<tr>
			<td>ascorbic acid</td>
			<td>Sigma-Aldrich, MO, USA</td>
			<td>A4544</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagen I</td>
			<td>Thermo Fisher Scientific</td>
			<td>A10483-01&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>fetal bovine serum</td>
			<td>Thermo Fisher Scientific</td>
			<td>10099141</td>
			<td></td>
		</tr>
		<tr>
			<td>homogenizer</td>
			<td>BiHeng&#160; Biotechnology, Shanghai, China</td>
			<td>SKSI</td>
			<td></td>
		</tr>
		<tr>
			<td>laser confocal fluorescence microscopy</td>
			<td>Carl&#160;Zeiss, Oberkochen, Germany</td>
			<td>LSM 800</td>
			<td></td>
		</tr>
		<tr>
			<td>Live/Dead Cell Imaging kit</td>
			<td>Thermo Fisher Scientific</td>
			<td>R37601</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel matrix</td>
			<td>Corning, NY, USA</td>
			<td>356234</td>
			<td></td>
		</tr>
		<tr>
			<td>MEM (10X), no glutamine</td>
			<td>Thermo Fisher Scientific</td>
			<td>21430079</td>
			<td></td>
		</tr>
		<tr>
			<td>paraformaldehyde</td>
			<td>Sigma-Aldrich, MO, USA</td>
			<td>158127</td>
			<td></td>
		</tr>
		<tr>
			<td>phosphate buffered saline</td>
			<td>Hyclone</td>
			<td>SH30256.FS</td>
			<td></td>
		</tr>
		<tr>
			<td>stereo microscope</td>
			<td>Carl&#160;Zeiss, Oberkochen, Germany</td>
			<td>Zeiss Axio ZOOM.V16</td>
			<td></td>
		</tr>
		<tr>
			<td>Trizol</td>
			<td>Thermo Fisher Scientific</td>
			<td>15596026</td>
			<td></td>
		</tr>
		<tr>
			<td>X-ray fluorescence</td>
			<td>EDAX, USA</td>
			<td>EAGLE III</td>
			<td></td>
		</tr>
		<tr>
			<td>&#946;-glycerophosphate</td>
			<td>Sigma-Aldrich, MO, USA</td>
			<td>G9422</td>
			<td></td>
		</tr>
	</tbody>
</table>

idg-sw3 cell culture in a three-dimensional extracellular matrix

ओस्टियोसाइट्स को नॉनप्रोलिफेरेटिव कोशिकाएं माना जाता है जो ओस्टियोब्लास्ट से टर्मिनल रूप से विभेदित होती हैं। अस्थि बाह्य मैट्रिक्स (ओस्टियोइड) में एम्बेडेड ओस्टियोब्लास्ट सेलुलर डेंड्राइट बनाने और प्रीओस्टियोसाइट्स में बदलने के लिए पीडीपीएन जीन को व्यक्त करते हैं। बाद में, प्रीओस्टियोसाइट्स मैट्रिक्स खनिज को बढ़ावा देने के लिए डीएमपी 1 जीन को व्यक्त करते हैं और इस तरह परिपक्व ओस्टियोसाइट्स में बदल जाते हैं। इस प्रक्रिया को ओस्टियोसाइटोजेनेसिस कहा जाता है। आईडीजी-एसडब्ल्यू 3 ओस्टियोसाइटोजेनेसिस के इन विट्रो अध्ययन के लिए एक प्रसिद्ध सेल लाइन है। कई पिछले तरीकों ने कोलेजन I को संवर्धन मैट्रिक्स के मुख्य या एकमात्र घटक के रूप में उपयोग किया है। हालांकि, कोलेजन I के अलावा, ऑस्टियोइड में एक जमीनी पदार्थ भी होता है, जो सेलुलर विकास, आसंजन और प्रवास को बढ़ावा देने में एक महत्वपूर्ण घटक है। इसके अलावा, मैट्रिक्स पदार्थ पारदर्शी है, जो कोलेजन आई-गठित जेल की पारदर्शिता को बढ़ाता है और इस प्रकार, इमेजिंग तकनीकों के माध्यम से डेंड्राइट गठन की खोज में सहायता करता है। इस प्रकार, यह पेपर आईडीजी-एसडब्ल्यू 3 अस्तित्व के लिए कोलेजन आई के साथ एक बाह्य मैट्रिक्स का उपयोग करके 3 डी जेल स्थापित करने के लिए एक प्रोटोकॉल का विवरण देता है। इस काम में, ऑस्टियोसाइटोजेनेसिस के दौरान डेंड्राइट गठन और जीन अभिव्यक्ति का विश्लेषण किया गया था। ओस्टोजेनिक कल्चर के 7 दिनों के बाद, एक व्यापक डेंड्राइट नेटवर्क स्पष्ट रूप से फ्लोरेसेंस कॉन्फोकल माइक्रोस्कोप के तहत देखा गया था। रियल-टाइम पीसीआर से पता चला कि पीडीपीएन और डीएमपी 1 के एमआरएनए स्तर लगातार 3 सप्ताह तक बढ़े। सप्ताह 4 में, स्टीरियोमाइक्रोस्कोप ने खनिज कणों से भरे एक अपारदर्शी जेल का खुलासा किया, जो एक्स-रे फ्लोरेसेंस (एक्सआरएफ) परख के अनुरूप था। इन परिणामों से संकेत मिलता है कि यह कल्चर मैट्रिक्स सफलतापूर्वक ओस्टियोब्लास्ट से परिपक्व ओस्टियोसाइट्स में संक्रमण की सुविधा प्रदान करता है।

मृत कोशिका धुंधला होने के बाद, कोशिकाओं को एक कॉन्फोकल लेजर माइक्रोस्कोप का उपयोग करके कल्पना की गई थी। सभी कोशिकाएं कैल्केन एएम-पॉजिटिव (हरा रंग) थीं, और क्षेत्र में लगभग कोई ईटीएचडी -1-पॉजिटिव कोशिकाए?...

Watch this Scientific Journal Video about IDG-SW3 Cell Culture in a Three-Dimensional Extracellular Matrix at JoVE.com

IDG-SW3 Cell Culture in a Three-Dimensional Extracellular Matrix

यहां, हम एक त्रि-आयामी (3 डी) बाह्य मैट्रिक्स में आईडीजी-एसडब्ल्यू 3 कोशिकाओं के संवर्धन के लिए एक प्रोटोकॉल प्रस्तुत करते हैं।

आईडीजी-एसडब्ल्यू 3 सेल कल्चर एक त्रि-आयामी बाह्य मैट्रिक्स में

Osteocytes are considered to be nonproliferative cells that are terminally differentiated from osteoblasts. Osteoblasts embedded in the bone extracellular ...

idg-sw3-cell-culture-in-a-three-dimensional-extracellular-matrix

Research

JoVE Journal

Bioengineering

Three-dimensional Optical-resolution Photoacoustic Microscopy

Optical microscopy, providing valuable insights at the cellular and organelle levels, has been widely recognized as an enabling biomedical technology. As the mainstays of in vivo three-dimensional (3-D) optical microscopy, single-/multi-photon fluorescence microscopy and optical coherence tomography (OCT) have demonstrated their extraordinary sensitivities to fluorescence and optical scattering contrasts, respectively. However, the optical absorption contrast of biological tissues, which encodes essential physiological/pathological information, has not yet been assessable. 
The emergence of biomedical photoacoustics has led to a new branch of optical microscopy optical-resolution photoacoustic microscopy (OR-PAM)1, where the optical irradiation is focused to the diffraction limit to achieve cellular1 or even subcellular2 level lateral resolution. As a valuable complement to existing optical microscopy technologies, OR-PAM brings in at least two novelties. First and most importantly, OR-PAM detects optical absorption contrasts with extraordinary sensitivity (i.e., 100%). Combining OR-PAM with fluorescence microscopy3 or with optical-scattering-based OCT4 (or with both) provides comprehensive optical properties of biological tissues. Second, OR-PAM encodes optical absorption into acoustic waves, in contrast to the pure optical processes in fluorescence microscopy and OCT, and provides background-free detection. The acoustic detection in OR-PAM mitigates the impacts of optical scattering on signal degradation and naturally eliminates possible interferences (i.e., crosstalks) between excitation and detection, which is a common problem in fluorescence microscopy due to the overlap between the excitation and fluorescence spectra. 
Unique for optical absorption imaging, OR-PAM has demonstrated broad biomedical applications since its invention, including, but not limited to, neurology5, 6, ophthalmology7, 8, vascular biology9, and dermatology10. In this video, we teach the system configuration and alignment of OR-PAM as well as the experimental procedures for in vivo functional microvascular imaging.

Optical-resolution photoacoustic microscopy (OR-PAM) is an emerging technology capable of imaging optical absorption contrasts in vivo with cellular resolution and sensitivity. Here, we provide a visualized instruction on the experimental protocols of OR-PAM, including system configuration, system alignment, typical in vivo experimental procedures, and functional imaging schemes.

तीन आयामी ऑप्टिकल संकल्प Photoacoustic माइक्रोस्कोपी

Optical microscopy, providing valuable insights at the cellular and organelle levels, has been widely recognized as an enabling biomedical technology. As ...

Live-cell Imaging of Migrating Cells Expressing Fluorescently-tagged Proteins in a Three-dimensional Matrix

Traditionally, cell migration has been studied on two-dimensional, stiff plastic surfaces. However, during important biological processes such as wound healing, tissue regeneration, and cancer metastasis, cells must navigate through complex, three-dimensional extracellular tissue. To better understand the mechanisms behind these biological processes, it is important to examine the roles of the proteins responsible for driving cell migration. Here, we outline a protocol to study the mechanisms of cell migration using the epithelial cell line (MDCK), and a three-dimensional, fibrous, self-polymerizing matrix as a model system. This optically clear extracellular matrix is easily amenable to live-cell imaging studies and better mimics the physiological, soft tissue environment. This report demonstrates a technique for directly visualizing protein localization and dynamics, and deformation of the surrounding three-dimensional matrix. 
Examination of protein localization and dynamics during cellular processes provides key insight into protein functions. Genetically encoded fluorescent tags provide a unique method for observing protein localization and dynamics. Using this technique, we can analyze the subcellular accumulation of key, force-generating cytoskeletal components in real-time as the cell maneuvers through the matrix. In addition, using multiple fluorescent tags with different wavelengths, we can examine the localization of multiple proteins simultaneously, thus allowing us to test, for example, whether different proteins have similar or divergent roles. Furthermore, the dynamics of fluorescently tagged proteins can be quantified using Fluorescent Recovery After Photobleaching (FRAP) analysis. This measurement assays the protein mobility and how stably bound the proteins are to the cytoskeletal network.
By combining live-cell imaging with the treatment of protein function inhibitors, we can examine in real-time the changes in the distribution of proteins and morphology of migrating cells. Furthermore, we also combine live-cell imaging with the use of fluorescent tracer particles embedded within the matrix to visualize the matrix deformation during cell migration. Thus, we can visualize how a migrating cell distributes force-generating proteins, and where the traction forces are exerted to the surrounding matrix. Through these techniques, we can gain valuable insight into the roles of specific proteins and their contributions to the mechanisms of cell migration.

Cellular processes such as cell migration have traditionally been studied on two-dimensional, stiff plastic surfaces. This report describes a technique for directly visualizing protein localization and analyzing protein dynamics in cells migrating in a more physiologically relevant, three-dimensional matrix.

लाइव सेल प्रवास के लिए जाते एक तीन आयामी मैट्रिक्स में प्रोटीन fluorescently टैग व्यक्त कोशिकाओं की इमेजिंग

Traditionally, cell migration has been studied on two-dimensional, stiff plastic surfaces. However, during important biological processes such as wound ...

Three-dimensional Cell Culture Model for Measuring the Effects of Interstitial Fluid Flow on Tumor Cell Invasion

The growth and progression of most solid tumors depend on the initial transformation of the cancer cells and their response to stroma-associated signaling in the tumor microenvironment 1. Previously, research on the tumor microenvironment has focused primarily on tumor-stromal interactions 1-2. However, the tumor microenvironment also includes a variety of biophysical forces, whose effects remain poorly understood. These forces are biomechanical consequences of tumor growth that lead to changes in gene expression, cell division, differentiation and invasion3. Matrix density 4, stiffness 5-6, and structure 6-7, interstitial fluid pressure 8, and interstitial fluid flow 8 are all altered during cancer progression.
Interstitial fluid flow in particular is higher in tumors compared to normal tissues 8-10. The estimated interstitial fluid flow velocities were measured and found to be in the range of 0.1-3 &mu;m s-1, depending on tumor size and differentiation 9, 11. This is due to elevated interstitial fluid pressure caused by tumor-induced angiogenesis and increased vascular permeability 12. Interstitial fluid flow has been shown to increase invasion of cancer cells 13-14, vascular fibroblasts and smooth muscle cells 15. This invasion may be due to autologous chemotactic gradients created around cells in 3-D 16 or increased matrix metalloproteinase (MMP) expression 15, chemokine secretion and cell adhesion molecule expression 17. However, the mechanism by which cells sense fluid flow is not well understood. In addition to altering tumor cell behavior, interstitial fluid flow modulates the activity of other cells in the tumor microenvironment. It is associated with (a) driving differentiation of fibroblasts into tumor-promoting myofibroblasts 18, (b) transporting of antigens and other soluble factors to lymph nodes 19, and (c) modulating lymphatic endothelial cell morphogenesis 20.
The technique presented here imposes interstitial fluid flow on cells in vitro and quantifies its effects on invasion (Figure 1). This method has been published in multiple studies to measure the effects of fluid flow on stromal and cancer cell invasion 13-15, 17. By changing the matrix composition, cell type, and cell concentration, this method can be applied to other diseases and physiological systems to study the effects of interstitial flow on cellular processes such as invasion, differentiation, proliferation, and gene expression.

Interstitial fluid flow is elevated in solid tumors and can modulate tumor cell invasion. Here we describe a technique to apply interstitial fluid flow to cells embedded in a matrix and then measure its effects on cell invasion. This technique can be easily adapted to study other systems.

ट्यूमर सेल आक्रमण पर बीचवाला द्रव प्रवाह के प्रभाव को मापने के लिए तीन आयामी सेल संस्कृति मॉडल

The growth and progression of most solid tumors depend on the initial transformation of the cancer cells and their response to stroma-associated signaling ...

Analysis of Cell Migration within a Three-dimensional Collagen Matrix

The ability to migrate is a hallmark of various cell types and plays a crucial role in several physiological processes, including&#160;embryonic development, wound healing, and immune responses. However, cell migration is also a key mechanism in cancer enabling these cancer cells to detach from the primary tumor to start metastatic spreading. Within the past years various cell migration assays have been developed to analyze the migratory behavior of different cell types. Because the locomotory behavior of cells markedly differs between a two-dimensional (2D) and three-dimensional (3D) environment it can be assumed that the analysis of the migration of cells that are embedded within a 3D environment would yield in more significant cell migration data. The advantage of the described 3D collagen matrix migration assay is that cells are embedded within a physiological 3D network of collagen fibers representing the major component of the extracellular matrix. Due to time-lapse video microscopy real cell migration is measured allowing the determination of several migration parameters as well as their alterations in response to pro-migratory factors or inhibitors. Various cell types could be analyzed using this technique, including lymphocytes/leukocytes, stem cells, and tumor cells. Likewise, also cell clusters or spheroids could be embedded within the collagen matrix concomitant with analysis of the emigration of single cells from the cell cluster/ spheroid into the collagen lattice. We conclude that the 3D collagen matrix migration assay is a versatile method to analyze the migration of cells within a physiological-like 3D environment.

Cell migration is a biological phenomenon that is involved in a plethora of physiological, such as wound healing and immune responses, and pathophysiological processes, like cancer. The 3D-collagen matrix migration assay is a versatile tool to analyze the migratory properties of different cell types within in a 3D physiological-like environment.

एक तीन आयामी मैट्रिक्स कोलेजन के भीतर सेल प्रवास का विश्लेषण

The ability to migrate is a hallmark of various cell types and plays a crucial role in several physiological processes, including&#160;embryonic ...

Generation of a Three-dimensional Full Thickness Skin Equivalent and Automated Wounding

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models reflect the human wound situation better than animal models. Although two-dimensional models are widely used to investigate processes such as cellular migration and proliferation, models that are more complex are required to gain a deeper knowledge about wound healing. Besides a suitable model system, the generation of precise and reproducible wounds is crucial to ensure comparable results between different test runs. In this study, the generation of a three-dimensional full thickness skin equivalent to study wound healing is shown. The dermal part of the models is comprised of human dermal fibroblast embedded in a rat-tail collagen type I hydrogel. Following the inoculation with human epidermal keratinocytes and consequent culture at the air-liquid interface, a multilayered epidermis is formed on top of the models. To study the wound healing process, we additionally developed an automated wounding device, which generates standardized wounds in a sterile atmosphere.

The goal of this protocol is to build up a three-dimensional full thickness skin equivalent, which resembles natural skin. With a specifically constructed automated wounding device, precise and reproducible wounds can be generated under maintenance of sterility.

एक तीन आयामी पूर्ण मोटाई त्वचा की पीढ़ी के बराबर है और स्वचालित लोग घायल हो गए

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models ...

Epithelial Cell Repopulation and Preparation of Rodent Extracellular Matrix Scaffolds for Renal Tissue Development

This protocol details the generation of acellular, yet biofunctional, renal extracellular matrix (ECM) scaffolds that are useful as small-scale model substrates for organ-scale tissue development. Sprague Dawley rat kidneys are cannulated by inserting a catheter into the renal artery and perfused with a series of low-concentration detergents (Triton X-100 and sodium dodecyl sulfate (SDS)) over 26 hr to derive intact, whole-kidney scaffolds with intact perfusable vasculature, glomeruli, and renal tubules. Following decellularization, the renal scaffold is placed inside a custom-designed perfusion bioreactor vessel, and the catheterized renal artery is connected to a perfusion circuit consisting of: a peristaltic pump; tubing; and optional probes for pH, dissolved oxygen, and pressure. After sterilizing the scaffold with peracetic acid and ethanol, and balancing the pH (7.4), the kidney scaffold is prepared for seeding via perfusion of culture medium within a large-capacity incubator maintained at 37 &#176;C and 5% CO2. Forty million renal cortical tubular epithelial (RCTE) cells are injected through the renal artery, and rapidly perfused through the scaffold under high flow (25 ml/min) and pressure (~230 mmHg) for 15 min before reducing the flow to a physiological rate (4 ml/min). RCTE cells primarily populate the tubular ECM niche within the renal cortex, proliferate, and form tubular epithelial structures over seven days of perfusion culture. A 44 &#181;M resazurin solution in culture medium is perfused through the kidney for 1 hr during medium exchanges to provide a fluorometric, redox-based metabolic assessment of cell viability and proliferation during tubulogenesis. The kidney perfusion bioreactor permits non-invasive sampling of medium for biochemical assessment, and multiple inlet ports allow alternative retrograde seeding through the renal vein or ureter. These protocols can be used to recellularize kidney scaffolds with a variety of cell types, including vascular endothelial, tubular epithelial, and stromal fibroblasts, for rapid evaluation within this system.

This protocol describes decellularization of Sprague Dawley rat kidneys by antegrade perfusion of detergents through the vasculature, producing acellular renal extracellular matrices that serve as templates for repopulation with human renal epithelial cells. Recellularization and use of the resazurin perfusion assay to monitor growth is performed within specially-designed perfusion bioreactors.

उपकला सेल repopulation और गुर्दे ऊतकों के विकास के लिए कृंतक बाह्य मैट्रिक्स scaffolds के तैयारी

This protocol details the generation of acellular, yet biofunctional, renal extracellular matrix (ECM) scaffolds that are useful as small-scale model ...

Engineering Three-dimensional Epithelial Tissues Embedded within Extracellular Matrix

The architecture of branched organs such as the lungs, kidneys, and mammary glands arises through the developmental process of branching morphogenesis, which is regulated by a variety of soluble and physical signals in the microenvironment. Described here is a method created to study the process of branching morphogenesis by forming engineered three-dimensional (3D) epithelial tissues of defined shape and size that are completely embedded within an extracellular matrix (ECM). This method enables the formation of arrays of identical tissues and enables the control of a variety of environmental factors, including tissue geometry, spacing, and ECM composition. This method can also be combined with widely used techniques such as traction force microscopy (TFM) to gain more information about the interactions between cells and their surrounding ECM. The protocol can be used to investigate a variety of cell and tissue processes beyond branching morphogenesis, including cancer invasion.

This manuscript describes a soft lithography-based technique to engineer uniform arrays of three-dimensional (3D) epithelial tissues of defined geometry surrounded by extracellular matrix. This method is amenable to a wide variety of cell types and experimental contexts and allows for high-throughput screening of identical replicates.

इंजीनियरिंग तीन आयामी उपकला ऊतकों कोशिकी मैट्रिक्स के भीतर एम्बेडेड

The architecture of branched organs such as the lungs, kidneys, and mammary glands arises through the developmental process of branching morphogenesis, ...

Tunable Hydrogels from Pulmonary Extracellular Matrix for 3D Cell Culture

Here we present a method for establishing multiple component cell culture hydrogels for in vitro lung cell culture. Beginning with healthy en bloc lung tissue from porcine, rat, or mouse, the tissue is perfused and submerged in subsequent chemical detergents to remove the cellular debris. Histological comparison of the tissue before and after processing confirms removal of over 95% of double stranded DNA and alpha galactosidase staining suggests the majority of cellular debris is removed. After decellularization, the tissue is lyophilized and then cryomilled into a powder. The matrix powder is digested for 48 hr in an acidic pepsin digestion solution and then neutralized to form the pregel solution. Gelation of the pregel solution can be induced by incubation at 37 &#176;C and can be used immediately following neutralization or stored at 4 &#176;C for up to two weeks. Coatings can be formed using the pregel solution on a non-treated plate for cell attachment. Cells can be suspended in the pregel prior to self-assembly to achieve a 3D culture, plated on the surface of a formed gel from which the cells can migrate through the scaffold, or plated on the coatings. Alterations to the strategy presented can impact gelation temperature, strength, or protein fragment sizes. Beyond hydrogel formation, the hydrogel stiffness may be increased using genipin.

This is a method to create a 3-dimensional cell culture scaffold from pulmonary extracellular matrix. Intact lung is processed into hydrogels that can support the growth of cells in three-dimensions.

3 डी सेल संस्कृति के लिए पल्मोनरी बाह्य मैट्रिक्स से Tunable Hydrogels

Here we present a method for establishing multiple component cell culture hydrogels for in vitro lung cell culture. Beginning with healthy en bloc lung ...

Fabrication of Extracellular Matrix-derived Foams and Microcarriers as Tissue-specific Cell Culture and Delivery Platforms

Cell function is mediated by interactions with the extracellular matrix (ECM), which has complex tissue-specific composition and architecture. The focus of this article is on the methods for fabricating ECM-derived porous foams and microcarriers for use as biologically-relevant substrates in advanced 3D in vitro cell culture models or as pro-regenerative scaffolds and cell delivery systems for tissue engineering and regenerative medicine. Using decellularized tissues or purified insoluble collagen as a starting material, the techniques can be applied to synthesize a broad array of tissue-specific bioscaffolds with customizable geometries. The approach involves mechanical processing and mild enzymatic digestion to yield an ECM suspension that is used to fabricate the three-dimensional foams or microcarriers through controlled freezing and lyophilization procedures. These pure ECM-derived scaffolds are highly porous, yet stable without the need for chemical crosslinking agents or other additives that may negatively impact cell function. The scaffold properties can be tuned to some extent by varying factors such as the ECM suspension concentration, mechanical processing methods, or synthesis conditions. In general, the scaffolds are robust and easy to handle, and can be processed as tissues for most standard biological assays, providing a versatile and user-friendly 3D cell culture platform that mimics the native ECM composition. Overall, these straightforward methods for fabricating customized ECM-derived foams and microcarriers may be of interest to both biologists and biomedical engineers as tissue-specific cell-instructive platforms for in vitro and in vivo applications.

The tissue-specific extracellular matrix (ECM) is a key mediator of cell function. This article describes methods for synthesizing pure ECM-derived foams and microcarriers that are stable in culture without the need for chemical crosslinking for applications in advanced 3D in vitro cell culture models or as pro-regenerative bioscaffolds.

बाह्य मैट्रिक्स व्युत्पन्न foams और ऊतक विशेष सेल संस्कृति और प्रसव के प्लेटफार्म के रूप में Microcarriers का निर्माण

Cell function is mediated by interactions with the extracellular matrix (ECM), which has complex tissue-specific composition and architecture. The focus ...

Fabricating a Kidney Cortex Extracellular Matrix-Derived Hydrogel

Extracellular matrix (ECM) provides important biophysical and biochemical cues to maintain tissue homeostasis. Current synthetic hydrogels offer robust mechanical support for in vitro cell culture but lack the necessary protein and ligand composition to elicit physiological behavior from cells. This manuscript describes a fabrication method for a kidney cortex ECM-derived hydrogel with proper mechanical robustness and supportive biochemical composition. The hydrogel is fabricated by mechanically homogenizing and solubilizing decellularized human kidney cortex ECM. The matrix preserves native kidney cortex ECM protein ratios while also enabling gelation to physiological mechanical stiffnesses. The hydrogel serves as a substrate upon which kidney cortex-derived cells can be maintained under physiological conditions. Furthermore, the hydrogel composition can be manipulated to model a diseased environment which enables the future study of kidney diseases.

Here we present a protocol to fabricate a kidney cortex extracellular matrix-derived hydrogel to retain the native kidney extracellular matrix (ECM) structural and biochemical composition. The fabrication process and its applications are described. Finally, a perspective on using this hydrogel to support kidney-specific cellular and tissue regeneration and bioengineering is discussed.

एक गुर्दा प्रांतस्था Extracellular मैट्रिक्स-व्युत्पंन Hydrogel

Extracellular matrix (ECM) provides important biophysical and biochemical cues to maintain tissue homeostasis. Current synthetic hydrogels offer robust ...