<meta charset="utf8"></head><body>היווצרות וחיבור של קרומי משי FN לתוספות של תרבית תאים

<ol>
	<li>Kumar, P., Kedaria, D., Mahapatra, C., Mohandas, M., Chatterjee, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+designer+cell+culture+insert+with+a+nanofibrous+membrane+toward+engineering+an+epithelial+tissue+model+validated+by+cellular+nanomechanics.">A designer cell culture insert with a nanofibrous membrane toward engineering an epithelial tissue model validated by cellular nanomechanics.</a> Nanoscale Adv. 3 (16), 4714-4725 (2021).</li><li>Wang, Y., 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=Ultrathin+and+handleable+nanofibrous+net+as+a+novel+biomimetic+basement+membrane+material+for+endothelial+barrier+formation.">Ultrathin and handleable nanofibrous net as a novel biomimetic basement membrane material for endothelial barrier formation.</a> Colloids Surf B Biointerfaces. 219, 112775 (2022).</li><li>Jain, P., Rauer, S. B., M&#246;ller, M., Singh, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mimicking+the+natural+basement+membrane+for+advanced+tissue+engineering.">Mimicking the natural basement membrane for advanced tissue engineering.</a> Biomacromolecules. 23 (8), 3081-3103 (2022).</li><li>Eom, S., Park, S. M., Han, S. J., Kim, J. W., Kim, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=One-step+fabrication+of+a+tunable+nanofibrous+well+insert+via+electrolyte-assisted+electrospinning.">One-step fabrication of a tunable nanofibrous well insert via electrolyte-assisted electrospinning.</a> RSC Advances. 7 (61), 38300-38306 (2017).</li><li>Kim, D., Eom, S., Park, S. M., Hong, H., Kim, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+collagen+gel-coated,+aligned+nanofiber+membrane+for+enhanced+endothelial+barrier+function.">A collagen gel-coated, aligned nanofiber membrane for enhanced endothelial barrier function.</a> Sci Rep. 9 (1), 1-11 (2019).</li><li>Ebrahim, N. 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=Porous+honeycomb+film+membranes+enhance+endothelial+barrier+integrity+in+human+vascular+wall+bilayer+model+compared+to+standard+track-etched+membranes.">Porous honeycomb film membranes enhance endothelial barrier integrity in human vascular wall bilayer model compared to standard track-etched membranes.</a> J Biomed Mater Res A. 111 (5), 701-713 (2023).</li><li>Zakharova, 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=Transwell-integrated+2+&#181;m+thick+transparent+polydimethylsiloxane+membranes+with+controlled+pore+sizes+and+distribution+to+model+the+blood-brain+barrier.">Transwell-integrated 2 &#181;m thick transparent polydimethylsiloxane membranes with controlled pore sizes and distribution to model the blood-brain barrier.</a> Advanced Materials Technologies. 6 (12), 2100138 (2021).</li><li>Park, S., 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=Use+of+elastic,+porous,+and+ultrathin+co-culture+membranes+to+control+the+endothelial+barrier+function+via+cell+alignment.">Use of elastic, porous, and ultrathin co-culture membranes to control the endothelial barrier function via cell alignment.</a> Advanced Functional Materials. 31 (9), 2008172 (2021).</li><li>Youn, J., 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=Facile+and+adhesive-free+method+for+bonding+nanofiber+membrane+onto+thermoplastic+polystyrene+substrate+to+fabricate+3D+cell+culture+platforms.">Facile and adhesive-free method for bonding nanofiber membrane onto thermoplastic polystyrene substrate to fabricate 3D cell culture platforms.</a> Materials Today Bio. 20, 100648 (2023).</li><li>Liu, H., 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=Electrohydrodynamic+jet-printed+ultrathin+polycaprolactone+scaffolds+mimicking+Bruch&#8217;s+membrane+for+retinal+pigment+epithelial+tissue+engineering.">Electrohydrodynamic jet-printed ultrathin polycaprolactone scaffolds mimicking Bruch&#8217;s membrane for retinal pigment epithelial tissue engineering.</a> Int J Bioprint. 8 (3), 550 (2022).</li><li>Dogan, A. A., Dufva, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Customized+3D-printed+stackable+cell+culture+inserts+tailored+with+bioactive+membranes.">Customized 3D-printed stackable cell culture inserts tailored with bioactive membranes.</a> Sci Rep. 12 (1), 1-12 (2022).</li><li>Rohde, F., Danz, K., Jung, N., Wagner, S., Windbergs, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrospun+scaffolds+as+cell+culture+substrates+for+the+cultivation+of+an+in+vitro+blood&#8211;brain+barrier+model+using+human+induced+pluripotent+stem+cells.">Electrospun scaffolds as cell culture substrates for the cultivation of an in vitro blood&#8211;brain barrier model using human induced pluripotent stem cells.</a> Pharmaceutics. 14 (6), 1308 (2022).</li><li>Slater, S. C., 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+in+vitro+model+of+the+glomerular+capillary+wall+using+electrospun+collagen+nanofibres+in+a+bioartificial+composite+basement+membrane.">An in vitro model of the glomerular capillary wall using electrospun collagen nanofibres in a bioartificial composite basement membrane.</a> PLoS ONE. 6 (6), e20802 (2011).</li><li>Harris, T. I., 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=Utilizing+recombinant+spider+silk+proteins+to+develop+a+synthetic+Bruch&#8217;s+membrane+for+modeling+the+retinal+pigment+epithelium.">Utilizing recombinant spider silk proteins to develop a synthetic Bruch&#8217;s membrane for modeling the retinal pigment epithelium.</a> ACS Biomater Sci Eng. 5 (8), 4023-4036 (2019).</li><li>Hjelm, L. C., Hedhammar, M., L&#246;fblom, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+blood&#8211;brain+barrier+model+based+on+recombinant+spider+silk+protein+nanomembranes+for+evaluation+of+transcytosis+capability+of+biomolecules.">In blood&#8211;brain barrier model based on recombinant spider silk protein nanomembranes for evaluation of transcytosis capability of biomolecules.</a> Biochem Biophys Res Commun. 669, 77-84 (2023).</li><li>Gustafsson, L., 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=Recombinant+spider+silk+forms+tough+and+elastic+nanomembranes+that+are+protein-permeable+and+support+cell+attachment+and+growth.">Recombinant spider silk forms tough and elastic nanomembranes that are protein-permeable and support cell attachment and growth.</a> Advanced Functional Materials. 30 (40), 2002982 (2020).</li><li>Tasiopoulos, C. P., Gustafsson, L., Van Der Wijngaart, W., Hedhammar, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibrillar+nanomembranes+of+recombinant+spider+silk+protein+support+cell+co-culture+in+an+in+vitro+blood+vessel+wall+model.">Fibrillar nanomembranes of recombinant spider silk protein support cell co-culture in an in vitro blood vessel wall model.</a> ACS Biomater Sci Eng. 7 (7), 3332-3339 (2021).</li><li>Marino, A., Baronio, M., Buratti, U., Mele, E., Ciofani, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Porous+optically+transparent+cellulose+acetate+scaffolds+for+biomimetic+blood-brain+barrier+in+vitro+models.">Porous optically transparent cellulose acetate scaffolds for biomimetic blood-brain barrier in vitro models.</a> Front Bioeng Biotechnol. 9, 630063 (2021).</li><li>Schramm, R. A., Koslow, M. H., Nelson, D. A., Larsen, M., Castracane, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+impedance+biosensor+for+measurement+of+trans-epithelial+resistance+in+cells+cultured+on+nanofiber+scaffolds.">A novel impedance biosensor for measurement of trans-epithelial resistance in cells cultured on nanofiber scaffolds.</a> Biosensors. 7 (3), 35 (2017).</li><li>Qi, D., 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=Establishment+of+a+human+iPSC-+and+nanofiber-based+microphysiological+blood-brain+barrier+system.">Establishment of a human iPSC- and nanofiber-based microphysiological blood-brain barrier system.</a> ACS Appl Mater Interfaces. 10 (26), 21825-21835 (2018).</li><li>Rickabaugh, E., Weatherston, D., Harris, T. I., Jones, J. A., Vargis, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+a+biomimetic+in+vitro+model+of+Bruch&#8217;s+membrane+using+hagfish+slime+intermediate+filament+proteins.">Engineering a biomimetic in vitro model of Bruch&#8217;s membrane using hagfish slime intermediate filament proteins.</a> ACS Biomater Sci Eng. 9 (8), 5051-5061 (2023).</li><li>Choi, J. W., Youn, J., Kim, D. S., Park, T. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+iPS-derived+blood-brain+barrier+model+exhibiting+enhanced+barrier+properties+empowered+by+engineered+basement+membrane.">Human iPS-derived blood-brain barrier model exhibiting enhanced barrier properties empowered by engineered basement membrane.</a> Biomaterials. 293, 121983 (2023).</li><li>Widhe, M., Shalaly, N. D., Hedhammar, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+fibronectin+mimetic+motif+improves+integrin+mediated+cell+biding+to+recombinant+spider+silk+matrices.">A fibronectin mimetic motif improves integrin mediated cell biding to recombinant spider silk matrices.</a> Biomaterials. 74, 256-266 (2016).</li><li>Johansson, U., 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=Assembly+of+functionalized+silk+together+with+cells+to+obtain+proliferative+3D+cultures+integrated+in+a+network+of+ECM-like+microfibers.">Assembly of functionalized silk together with cells to obtain proliferative 3D cultures integrated in a network of ECM-like microfibers.</a> Sci Rep. 9 (1), 1-13 (2019).</li><li>Widhe, M., Johansson, U., Hillerdahl, C. O., Hedhammar, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recombinant+spider+silk+with+cell+binding+motifs+for+specific+adherence+of+cells.">Recombinant spider silk with cell binding motifs for specific adherence of cells.</a> Biomaterials. 34 (33), 8223-8224 (2013).</li></ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>CHEMICALS</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 488</td>
			<td>Invitrogen; Thermo Fisher Scientific</td>
			<td>A-21121</td>
			<td>Goat anti-mouse, Dilution 1:500</td>
		</tr>
		<tr>
			<td>Alexa Fluor 488 Phalloidin</td>
			<td>Invitrogen; Thermo Fisher Scientific</td>
			<td>A12379</td>
			<td>Dilution 1:400</td>
		</tr>
		<tr>
			<td>anti-ZO-1 (1A12) antibody</td>
			<td>Invitrogen; Thermo Fisher Scientific</td>
			<td>&#160;33-9100</td>
			<td>Mouse anti-human, Monoclonal, Dilution 1:200</td>
		</tr>
		<tr>
			<td>Dextran, Alexa Fluor 680; 3,000 MW, Anionic</td>
			<td>Invitrogen; Thermo Fisher Scientific</td>
			<td>D34681</td>
			<td>Diluted 2,5% (w/v) in 200 ul of culture medium</td>
		</tr>
		<tr>
			<td>DMEM/F-12</td>
			<td>Gibco; Thermo Fisher Scientific</td>
			<td>31330095</td>
			<td>Supplemented with 5% v/v FBS and 1% v/v Penicillin-Streptomycin</td>
		</tr>
		<tr>
			<td>Ethanol&#160;</td>
			<td>Solveco</td>
			<td>1326</td>
			<td>70% (CAS-no 64-17-5)</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum, qualified, heat inactivated, United States</td>
			<td>Gibco; Thermo Fisher Scientific</td>
			<td>16140071</td>
			<td></td>
		</tr>
		<tr>
			<td>FN-silk</td>
			<td>Spiber technologies AB</td>
			<td></td>
			<td>Store at -80 &#176;C</td>
		</tr>
		<tr>
			<td>Isopropanol, EMPARTA ACS analytical reagent</td>
			<td>Supelco</td>
			<td>1096342511</td>
			<td>&#8805;99.5% (CAS-no 67-63-0)</td>
		</tr>
		<tr>
			<td>Live/Dead Viability/Cytotoxicity Kit</td>
			<td>Invitrogen; Thermo Fisher Scientific</td>
			<td>L3224</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS&#160;</td>
			<td>Swedish Veterinary Agency / Statens veterin&#228;rmedicinska anstalt</td>
			<td>992420</td>
			<td>&#160;without Ca and Mg, filtered</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin (10,000 U/mL)</td>
			<td>Gibco; Thermo Fisher Scientific</td>
			<td>11548876</td>
			<td></td>
		</tr>
		<tr>
			<td>MATERIAL</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dental LT Clear Resin</td>
			<td>Denthouse</td>
			<td>&#160;#DLCL-01</td>
			<td></td>
		</tr>
		<tr>
			<td>HaCaT cells</td>
			<td>CLS</td>
			<td>300493</td>
			<td></td>
		</tr>
		<tr>
			<td>Nunc Cell-Culture Treated Multidishes 24-well</td>
			<td>Fisher Scientific</td>
			<td>10604903</td>
			<td></td>
		</tr>
		<tr>
			<td>Nunc Cell-Culture Treated Multidishes 48-wel</td>
			<td>Fisher Scientific</td>
			<td>10644901</td>
			<td></td>
		</tr>
		<tr>
			<td>TC insert, for 24-well plates, PET, transparent</td>
			<td>Sarstedt</td>
			<td>83.3932.041</td>
			<td>pore size: 0.4 &#181;m</td>
		</tr>
		<tr>
			<td>ThinCert Cell Culture Inserts, translusent membrane (PET)</td>
			<td>Greiner</td>
			<td>662640</td>
			<td>pore size: 0.4 &#181;m</td>
		</tr>
		<tr>
			<td>EQUIPMENT</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>EVOM meter with chopsticks&#160;</td>
			<td>World Precision Instruments (WPI) Germany, GMBH</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Form 3B</td>
			<td>FormLabs</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Form Wash</td>
			<td>FormLabs</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Form Cure</td>
			<td>FormLabs</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Isotemp General Purpose Deluxe Water Baths</td>
			<td>Fisherbrand</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted fluorescence microscope Eclipse Ti</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted fluorescence microscope DMI6000 B</td>
			<td>Leica</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Laminar flow hood Ninosafe, class II</td>
			<td>Labolutions</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Midi CO2&#160;Incubator, 40 L</td>
			<td>Thermo Scientific</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

nanofibrillar basement membrane mimic made of recombinant functionalized spider silk in custom-made tissue culture inserts

In vitro models that can replicate tissue barriers have received increasing attention due to their applicability in testing novel therapeutics and facilitating the understanding of fundamental disease mechanisms1,2. To accurately recreate the native microenvironment, it is critical to recapitulate the function of the basement membrane (BM), a highly specialized extracellular matrix (ECM) complex. The BM exists in almost every tissue in the human body, where it provides support to endothelial and epithelial cells and separates them from underlying tissue2,3. Beyond providing physical support, the BM also regulates and maintains biochemical signals between the cells and surrounding tissue. These vital functions make it necessary to design scaffolds that resemble the structure, as well as the mechanical and functional characteristics of the native BM3.
One of the most common ways to mimic the BM in vitro today is through the use of commercially available tissue culture inserts (TC-inserts). These are essentially plastic cylinders with a permeable membrane that divides the chamber into apical and basolateral sides4,5. While easy to use, the membranes in commercial inserts are generally rigid, track-etched, and made from polymers such as poly(carbonate) (PC) and poly(ethylene terephthalate) (PET)3,4,6. They are flexible in terms of diameter, pore density, and size and can be coated to enhance cell adhesion but lack all other relevant features of the BM, such as comparable thickness6, interconnected porosity, fibrous architecture, and relevant elastic modulus3.
The standardization of TC inserts and their ease of use has inspired several groups, including ours, to replace the plastic membrane with a more in vivo-like counterpart (overviewed in Table 1). The materials used range from polymers such as Polydimethylsiloxane (PDMS)7, Poly(lactide-co-caprolactone) (PLCL)8, and polycaprolactone (PCL)4,9,10 to protein-based materials such as gelatin2,11,12, collagen5,13, and recombinant spider silk14,15,16,17. Membranes from these materials have been attached in various ways, both to commercially available inserts from which the membrane has been removed4,7,8,10,12,13,14,16,18,19,20,21, as well as to custom-designed inserts manufactured through 3D-printing1,11,15,17,20 or injection molding9,22. However, most of these still come far from resembling the native BM in terms of thickness, where the replicas range from hundreds11,18 down to a few micrometers5,10,14,21,22. Many of them also require complex formation and/or manual attachment methods1,7,13,14,18,19,21, making scaling and replication in other labs challenging.
Herein, we present a simple method to form and attach a silk membrane to inserts and show how to culture cells on either side of the membrane. The silk membranes are formed through self-assembly of the FN-4RepCT (FN-silk) protein at the liquid-air interface of a standing solution16,17. FN-silk is a recombinantly produced short version of Major Ampullulate Spidroin 1 from Euprosthenops australis, functionalized with an RGD motif derived from fibronectin23. It has been shown to assemble into fibrillar matrices that promote cell adhesion, growth, and migration15,16,17,23,24,25. The method for attachment of the membrane on the insert relies on spontaneous adhesion and has been found suitable for commercially available inserts from which the membrane has been removed16, as well as 3D-printed inserts from polylactic acid (PLA)17 and Dental LT15. This paper details how this method is used for inserts printed from Dental LT. After the FN-silk membranes have been attached to the inserts, they can, in essence, be treated like standard commercial tissue culture inserts. In short, we present a simple method to generate more relevant in vitro models of tissue barriers by replacing plastic membranes with a protein-based, FN-silk membrane.

<meta charset="utf8"></head><body>מחבר זרקור: שיפור מודלים של תרביות תאים חוץ גופיות עם קרומי משי עכביש פונקציונליים רקומביננטיים

חיקוי קרום מרתף ננופיברילרי עשוי ממשי עכביש פונקציונלי רקומביננטי בתוספות תרבית רקמה בהתאמה אישית

Our research focuses on using recombinantly produced functionalized spider silk proteins to create extracellular matrix models to create more in vivo-like conditions for the cells. We're hoping that by creating a more realistic condition for the cells we're growing, they will grow into tissue-like construct just like they would inside the body, resulting in more realistic models. So the main advantage with using our FN-silk membranes instead of the commercially available plastic PET membranes is that the user gets a membrane that actually resembles the native basement membrane in terms of structure, thickness, biological composition, and elasticity.By using a membrane that comes closer to mimicking the native basement membrane, we're able to establish more realistic in-vitro tissue models. This can speed up drug discovery and further reduce the need for animal testing. We will continue to investigate how the FN-silk membrane can further improve static in-vitro tissue models, and we will also incorporate the membrane into microfluidic chips in order to expose the cells to flow.This way we can further involve in-vitro models towards more in vivo-like conditions.

Replicating tissue barriers&#160;is critical for generating relevant in vitro models for evaluating novel therapeutics. Today, this is commonly done using ...

nanofibrillar-basement-membrane-mimic-made-recombinant-functionalized

Research

JoVE Journal

Bioengineering

Nanofibrillar Basement Membrane Mimic Made of Recombinant Functionalized Spider Silk in Custom-Made Tissue Culture Inserts

507 Views.  Spiber Technologies AB. המחקר שלנו מתמקד בשימוש בחלבוני משי עכביש פונקציונליים המיוצרים באופן רקומביננטי כדי ליצור מודלים של מטריצה חוץ-תאית כדי ליצור יותר תנאים דמויי vivo עבור התאים. אנו מקווים שעל ידי יצירת מצב מציאותי יותר עבור התאים שאנו מגדלים, הם יגדלו למבנה דמוי רקמה בדיו...

Video: מחבר זרקור: שיפור מודלים של תרביות תאים חוץ גופיות עם קרומי משי עכביש פונקציונליים רקומביננטיים

Replicating tissue barriers&#160;is critical for generating relevant in vitro models for evaluating novel therapeutics. Today, this is commonly done using tissue culture inserts with a plastic membrane, which generates an apical and a basal side. Besides providing support for the cells, these membranes come far from emulating their native counterpart, the basement membrane, which is a nanofibrillar, protein-based matrix. In this work, we show a simple way to considerably improve the biological relevance of the tissue culture inserts by replacing the plastic membrane with one made from a pure recombinant functionalized spider silk protein. The silk membrane forms through self-assembly and will spontaneously adhere to a membrane-free tissue culture insert, where it can provide support for cells. Custom-designed tissue culture inserts can be printed using a standard 3D printer, following the instructions provided in the protocol, or commercial ones can be purchased and used instead. This protocol shows how the culture system with silk membranes in inserts is set up and, subsequently, how the same cell culturing techniques that are used with traditional, commercially available inserts can be implemented.

Tissue culture inserts with plastic membranes are the golden standard in cell culture labs as permeable supports to establish cell layers and models of barrier tissues. Herein, we present a simple method to replace the plastic membrane with a more biologically relevant membrane made from a recombinant functionalized spider silk protein.

Cell Seeding of Recombinant Spider Silk Membranes for Improved In Vitro Barrier Models

To begin, remove the FN-silk vials from the minus 80 degrees Celsius freezer, and place them in dry ice for transportation. Bring the vials to a biological safety cabinet, and place them in a micro centrifuge tube rack. Upon thawing, pipette 550 microliters of silk solution into every second well of a 48-well plate.Place the lid on the plate, and place it inside a sterile box. Carefully transfer the box from the biological safety cabinet, and leave it in ambient conditions overnight. The next day, bring the box containing the plate with the FN-silk membranes and sterile inserts into the biological safety cabinet.Carefully lift the plate from the box, open the lid of the plate, and visually verify membrane formation by observing cloudiness in the wells. Using sterile tweezers, pick up one insert and guide it down onto the membrane until the handles touch the top of the well. Once all inserts have been lowered, place the lid on the plate, and return the plate to the sterile box.Allow the membranes to adhere to the inserts for two hours. Next, remove the plate from the box and open the lid. Using sterile tweezers, remove the insert from the well.Fill the insert with 100 microliters of DMEM/F12 for basal seeding, or 200 microliters for apical seeding. Transfer the insert into an empty well of a 24-well plate, and fill the well with one milliliter of DMEM/F12. After harvesting the desired number of human keratinocytes, using a P200 pipette, aspirate 20 to 50 microliters of cell suspension.Aim the pipette tip towards the center of the membrane and slowly press the pipette plunger, bringing the droplet into contact with the culture medium inside the insert. Once all membranes are transferred, move the plate in a figure eight pattern to distribute the cells evenly across the membranes. Incubate the culture at 37 degrees Celsius and 5%carbon dioxide.Using tweezers, lift the inserts out of the 24-well plate, leaving the culture medium in the wells. Then invert the insert so that the basal side faces upwards. Place the inverted insert in the Petri dish.Aspirate 20 microliters of the resuspended human keratinocyte cell suspension with a P200 pipette. Aim the pipette tip towards the center of the membrane, and slowly press the pipette plunger to form a droplet that falls onto the membrane. Place the lid on the Petri dish.Transfer the Petri dish and medium-containing 24-well plate to the incubator for 30 minutes. After incubation, bring the 24-well plate and Petri dish into the biological safety cabinet. Using tweezers, pick one insert and invert it so that the apical side of the membrane faces upwards and the basal side downwards.Using a P200 pipette, add 200 microliters of culture medium inside the insert. Place the insert back into a prefilled well of the 24-well plate. Culture the inserts at 37 degrees Celsius and 5%carbon dioxide.Keratinocytes cultured on the FN-silk membrane evenly covered the surface and showed cobblestone morphology on day one. Keratinocytes formed a confluent layer by day three, indicating epithelial function, and formed a network of tight junctions indicating they were assuming physiologic epithelial functions. High cell viability was observed across the FN-silk membranes after three days, with no significant difference in viability between the center and periphery.The FN-silk membrane system supported keratinocyte viability similar to or better than commercial PET membrane systems.