The authors thank Michael Garwood and Sam Stein for their helpful comments, and Kristen M. Maynard for assistance with manuscript preparation.

The authors have nothing to disclose.

58 - biyolojik maddelerin ve hücre terapileri için üretimi için memeli hücre üreten ürünlerin büyük ölçüde 55 kültür ve memeli hücreleri izlenmesini gerektirir. Dahası, bu uygulamalar tanımlanmış ve valide kültür koşulları için çağrı. Sadece bu gereksinimleri karşılamak olmaz araştırma teknolojilerini kullanarak hücrelerin hacminin artırılması. besin ve atık ürünlerin birikimi dalgalanmalara yol açması, Manuel ortam değişiklikleri hücre kalites...

Transplantasyon için uygulanabilir ve fonksiyonel insan hücrelerinin çok sayıda oluşturmak için, kültür koşullarının düzenlenmesi zorunludur. 3 - metabolik atıkların birikimi ile birlikte besinlerin tükenmesi, hücre ürününün 1 kalitesini azaltır yaşlanması ve metabolik değişikliklere önemli sebeplerindendir. Bu prosedür kültürünün süresi boyunca fizyolojik aralığında 4 glikoz düzenleyen ayarlanmış oran perfüzyon besleme sistemi ile birlikte karıştırılan bir biyoreaktör kullanarak küresellerde kültür memeli hücrelerinin bir yöntemi gösterir. Bu çalışmaların amacı için, fizyolojik aralığı 100 ila 200 mg / dl olarak tanımlandı. Aynı yöntemleri diğer besin ve laktat gibi metabolik atıkları regüle etmek için kullanılabilir. küçük hacimlerde statik kültürler (1-30 mi), genellikle experime hücre hatları korumak ve ayırt laboratuar ortamında kullanılanntal amaçlar. Hücre pasajı düzenli aralıklarla gerektiğinde komple ortam değişiklikleri ile gerçekleştirilir. En &quot;klasik&quot; kültür ortamı, besin sınırlamalar riski olmadan daha az sık ve orta değişiklikleri sağlamak için, yüksek glukoz konsantrasyonuna (bu çalışmalarda kullanılan DMEM 450 mg / dL) sahiptir. Ancak, bu toplu beslenme yöntemi hala, sık manipülasyon gerektiren hücre ortamında değişkenlik tanıtır ve kontaminasyon 5 riskini artırır - 9. Karıştırılan süspansiyon biyoreaktörler (SSB) daha iyi karışmasını sağlamak ve 3,10 işleme azalmış - 20, ama statik kültürler gibi, besin ve atık ürün düzeylerinde potansiyel zarar verici dalgalanmalar katkıda manuel orta değişiklikler gerektirir. SSB kültürlerin perfüzyon besleme sürekli infüzyon ve orta kaldırılması, fakat hücre büyümesi besin seviyelerindeki büyük değişikliklere göre bu sorunların bir sorun olmaya devam azaltır. düzeltilmiş besleme ra kullanımıTahmini hücre gereksinimlerine göre besin kullanım hesaplamalarından te hücre canlılığını optimize etmek için gerekli kararlı hücre ortamı sağlamak ve 21 işlev görebilir - 24. 25 özel pluripotent hücrelerin kültür ve genişleme için memeli hücrelerinin ölçeklenebilir SSB kültürler için yöntemleri tarif literatür büyük bir gövde var - diğerleri ile 32, adacık (beta) hücreleri 17,33,34 odaklanmış, ya da biyolojik ürünlerin 24 üretim, 35-38. Bu araştırılmıştır hücre tiplerinin bir çok sferoid kültürlerde yetiştirilebilir ve kullanılan hücre tipi için özel prosedürler sürekli bir besleme sisteminin uygulanması öncesinde optimize edilmelidir. 43 - Bu gösteri, bir perfüzyon besleme yöntemi bir karıştıran bioreaktör 39 sferoidler olarak yetiştirilen beta hücre hattı genişletmek için kullanıldı. Bu tarifnamede tarif edilen metod sağlarhedeflenen kültür koşullarını elde etmek için off-line glukoz ölçümlerine dayanarak oranı ayarlamaları beslenme basit uygulama. Bu yöntem ile besleme oranı ayarlama fizyolojik glukoz seviyesi artar, hücre verimleri gösterilir korumak için. Memeli hücreleri, enerji üretimi için, önemli bir besin, glikoz bağlıdır, bu yüzden, bu hücre hattının kullanılması bir çok kültürlenmiş memeli hücrelerine 44 bir modeli temsil etmektedir. Buna ek olarak, bu hat glukoz 45, kronik yüksek düzeyde duyarlı olan beta hücreleri, daha karmaşık örnek teşkil etmektedir. Bu çalışma için, β-TC6 hücreler, in vivo olarak Langerhans adacıklarında ortalama büyüklüğü yaklaşık kültür içinde parçacıklarının oluşturulması için bırakılmıştır. Perfüzyon biyoreaktör sistemi 17 - tüketimi glikoz ayarlanmış bir besleme oranı ile 19,21,46, canlılığı değişiklik olmaksızın, fizyolojik koşullar ve yüksek hücre verimi muhafaza ile sonuçlanmıştır.

<table border="0" cellpadding="0" cellspacing="0" width="840">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:271px;">Name of Reagent/ Equipment</td>
			<td style="width:249px;">Company</td>
			<td style="width:153px;">Catalog Number / Link</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">BTC-6 Cells</td>
			<td style="width:249px;">ATCC, Manassas, VA</td>
			<td style="width:153px;">CRL-11506</td>
			<td>Mouse Insulinoma cell line (adherent cell type)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">DPBS No CA, No Mg</td>
			<td style="width:249px;">Invitrogen, Carlsbad, CA</td>
			<td style="width:153px;">14190-144</td>
			<td><a href="https://www.lifetechnologies.com/order/catalog/product/14190144?ICID=search-14190144">https://www.lifetechnologies.com/order/catalog/product/14190144?ICID=search-14190144</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Dulbecco&#39;s Modified Eagles Medium</td>
			<td style="width:249px;">Invitrogen, Carlsbad, CA</td>
			<td style="width:153px;"></td>
			<td>See below for product numbers&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">DMEM High Glucose (500mM)</td>
			<td style="width:249px;">Invitrogen, Carlsbad, CA</td>
			<td>11965-092</td>
			<td><a href="http://www.lifetechnologies.com/order/catalog/product/11965092">http://www.lifetechnologies.com/order/catalog/product/11965092</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">DMEM Low Glucose (100mM)</td>
			<td style="width:249px;">Invitrogen, Carlsbad, CA</td>
			<td>11885-084</td>
			<td><a href="http://www.lifetechnologies.com/order/catalog/product/11885084%20(note%20that%20this%20medium%20already%20contains%20pyruvate)">http://www.lifetechnologies.com/order/catalog/product/11885084 (note that this medium already contains pyruvate)</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">L-gultamine</td>
			<td style="width:249px;">Invitrogen, Carlsbad, CA</td>
			<td style="width:153px;">25030081</td>
			<td><a href="http://www.lifetechnologies.com/order/catalog/product/25030081?ICID=search-product">http://www.lifetechnologies.com/order/catalog/product/25030081?ICID=search-product</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Sodium Pyruvate</td>
			<td style="width:249px;">Invitrogen, Carlsbad, CA</td>
			<td style="width:153px;">11360070</td>
			<td><a href="https://www.lifetechnologies.com/order/catalog/product/11360070?ICID=search-product">https://www.lifetechnologies.com/order/catalog/product/11360070?ICID=search-product</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Heat Inactivated Porcine Serum</td>
			<td style="width:249px;">Gibco - Life Technologies</td>
			<td style="width:153px;">10082147</td>
			<td><a href="http://www.lifetechnologies.com/order/catalog/product/10082147">http://www.lifetechnologies.com/order/catalog/product/10082147</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Trypsin-EDTA</td>
			<td style="width:249px;">Invitrogen, Carlsbad, CA</td>
			<td style="width:153px;">25200056</td>
			<td><a href="https://www.lifetechnologies.com/order/catalog/product/25200056?ICID=search-product">https://www.lifetechnologies.com/order/catalog/product/25200056?ICID=search-product</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">T-150 Tissue Culture Treated Flasks</td>
			<td style="width:249px;">Corning, Corning, NY</td>
			<td style="width:153px;">430825</td>
			<td><a href="http://catalog2.corning.com/LifeSciences/en-US/Shopping/ProductDetails.aspx?productid=430825(Lifesciences)&amp;categoryname=">http://catalog2.corning.com/LifeSciences/en-US/Shopping/ProductDetails.aspx?productid=430825(Lifesciences) 
			&#38;categoryname=</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NuAire Cell culture incubator</td>
			<td style="width:249px;">Princeton, MN</td>
			<td style="width:153px;"></td>
			<td>US Autoflow , Any water-jacketed CO2 regulating cell culture incubator could be used</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Centrifuge</td>
			<td style="width:249px;"></td>
			<td style="width:153px;"></td>
			<td>Sorvall RT 7 (Any similar benchtop centrifuge may be used)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Refrigerator</td>
			<td style="width:249px;"></td>
			<td style="width:153px;"></td>
			<td>Any laboratory refrigerator could be used (a small table-top version was used for these studies)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">1L Glass Bottle</td>
			<td style="width:249px;">Corning, Corning, NY</td>
			<td style="width:153px;">1395-1L</td>
			<td>Any vendor could be used http://catalog2.corning.com/LifeSciences/en-US/Shopping/ProductDetails.aspx?productid=1395-1L(Lifesciences) 
			&#38;categoryname=</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">2L Glass Bottle</td>
			<td style="width:249px;">Corning, Corning, NY</td>
			<td style="width:153px;">1395-2L</td>
			<td>Any vendor could be used</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">250 ml stirred bioreactors&#160;</td>
			<td style="width:249px;">Corning, Corning, NY</td>
			<td style="width:153px;">4500-250</td>
			<td><a href="http://catalog2.corning.com/LifeSciences/en-US/Shopping/ProductDetails.aspx?productid=4500-250(Lifesciences)&amp;categoryname=">http://catalog2.corning.com/LifeSciences/en-US/Shopping/ProductDetails.aspx?productid=4500-250(Lifesciences) 
			&#38;categoryname=</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Stir Plate</td>
			<td style="width:249px;">Fisher Scientific</td>
			<td style="width:153px;">11-496-104A</td>
			<td>Any incubator safe stir-plate can be used, any vendor</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:271px;">Tissue Culture Dishes 100mm Diameter</td>
			<td style="width:249px;">Nunc, Rochester, NY (Fisher Scientific)</td>
			<td style="width:153px;">1256598&#160;</td>
			<td>Any vendor could be used (ordered through Fisher Sci)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">FALCON 50 ml Conical Tubes</td>
			<td style="width:249px;">Falcon, San Jose, CA</td>
			<td style="width:153px;">1256598</td>
			<td>Any vendor could be used</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Delran Plastic Used for Custom Parts</td>
			<td style="width:249px;">McMaster Carr</td>
			<td style="width:153px;">Various</td>
			<td>Any material of choice could be used, but Deran is chosen because it is autoclave safe, non-reactive, and easy to machine, http://www.mcmaster.com/#acetal-homopolymer-sheets/=rjrcac</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Stainless Steel Pipe for custom lids</td>
			<td style="width:249px;">McMaster Carr</td>
			<td style="width:153px;">Various</td>
			<td>Any vendor could be used, http://www.mcmaster.com/#standard-stainless-steel-tubing/=rjrd91</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:271px;">Custom Modified Delran Bioreactor Lids for Continuous Feeding</td>
			<td style="width:249px;">Custom made&#160;</td>
			<td style="width:153px;"></td>
			<td>Not aware of any vendors producing a similar product</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:271px;">Custom Modified Glass Bottle Lids for Continuous feeding</td>
			<td style="width:249px;">Custom made&#160;</td>
			<td style="width:153px;"></td>
			<td>Some vendors (eg. Fischer Sci, Corning) make similar products in the links below</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Masterflex Digital Peristaltic Pump</td>
			<td style="width:249px;">Cole Parmer, Vernon Hills, IL</td>
			<td style="width:153px;">EW-77919-25</td>
			<td>Any precision peristaltic pump could be used, http://www.coleparmer.com/Product/L_S_Eight_Channel_Four_Roller_ 
			Cartridge_Pump_System_115_230 
			_VAC/EW-77919-25</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">PVDF Tubing Connectors (various)</td>
			<td style="width:249px;">Cole Parmer, Vernon Hills, IL</td>
			<td style="width:153px;">see link</td>
			<td>Any vendor could be used, http://www.coleparmer.com/Category/Cole_Parmer_PVDF_Premium 
			_Luer_Fittings/55889</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Pharmed BPT Tubing L/S 16</td>
			<td style="width:249px;">Cole Parmer, Vernon Hills, IL</td>
			<td style="width:153px;">WU-06508-16</td>
			<td>Any vendor could be used, http://www.coleparmer.com/Product/Masterflex_PharMed_BPT_Tubing 
			_L_S_13_25/WU-06508-16</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Pharmed BPT Tubing L/S 14</td>
			<td style="width:249px;">Cole Parmer, Vernon Hills, IL</td>
			<td style="width:153px;">WU-06508-14</td>
			<td>Any vendor could be used, http://www.coleparmer.com/Product/Masterflex_PharMed_BPT_Tubing 
			_L_S_13_25/WU-06508-14</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Pharmed BPT Tubing L/S 13</td>
			<td style="width:249px;">Cole Parmer, Vernon Hills, IL</td>
			<td style="width:153px;">WU-06508-13</td>
			<td>Any vendor could be used, http://www.coleparmer.com/Product/Masterflex_PharMed_BPT_Tubing 
			_L_S_13_25/WU-06508-13</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:271px;">Millipore Millex GP PES membrane 0.22ul sterile syringe filter (used for venting, and medium filtration)</td>
			<td style="width:249px;">Fisher Scientific</td>
			<td style="width:153px;">SLGP033RS</td>
			<td>Any vendor could be used</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">25ml Graduated Pipette</td>
			<td style="width:249px;">Fisher Scientific</td>
			<td style="width:153px;">13-678-11</td>
			<td>Any vendor could be used, and various sizes may be used</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Pipetter</td>
			<td style="width:249px;">Fisher Scientific</td>
			<td style="width:153px;">13-681-15E</td>
			<td>Any vendor, or similar product could be used</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Hemocytometer</td>
			<td style="width:249px;">Fisher Scientific</td>
			<td style="width:153px;">02-671-6</td>
			<td>Any vendor, or similar product could be used</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Trypan Blue</td>
			<td style="width:249px;">Gibco - Life Technologies</td>
			<td style="width:153px;">15250-061</td>
			<td>Any vendor, or similar product could be used, https://www.lifetechnologies.com/order/catalog/product/15250061</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Inverted Light Microscope</td>
			<td style="width:249px;">Leica</td>
			<td style="width:153px;"></td>
			<td>Any vendor, or similar product could be used</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">One Touch Ultra Blood Glucose Meter</td>
			<td style="width:249px;">Fisher Scientific</td>
			<td style="width:153px;">22-029-293&#160;</td>
			<td>Any vendor, or similar product could be used (eg. Bayer)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">One Touch Ultra-Strips</td>
			<td style="width:249px;">Fisher Scientific</td>
			<td style="width:153px;">22-029-292&#160;</td>
			<td>Any vendor, or similar product could be used (eg. Bayer)</td>
		</tr>
	</tbody>
</table>

nutrient regulation by continuous feeding for large-scale expansion of mammalian cells in spheroids

In this demonstration, spheroids formed from the &#946;-TC6 insulinoma cell line were cultured as a model of manufacturing a mammalian islet cell product to demonstrate how regulating nutrient levels can improve cell yields. In previous studies, bioreactors facilitated increased culture volumes over static cultures, but no increase in cell yields were observed. Limitations in key nutrients such as glucose, which were consumed between batch feedings, can lead to limitations in cell expansion. Large fluctuations in glucose levels were observed, despite the increase in glucose concentrations in the media. The use of continuous feeding systems eliminated fluctuations in glucose levels, and improved cell growth rates when compared with batch fed static and SSB culture methods. Additional increases in growth rates were observed by adjusting the feed rate based on calculated nutrient consumption, which allowed the maintenance of physiological glucose over three weeks in culture. This method can also be adapted for other cell types.

Orta Glukoz Düzeyleri ve dalgalanmalar Standart SSB Kültürlerde Hücre Genişleme sınırla Glikoz düzeyleri kültür dönemi 3 boyunca statik kültürler ve SSB kültürlerde dalgalanma. Bu dalgalanmalar, 21 günlük kültür döneminde, hücre sayısı arttıkça yoğunlaştırmak ve statik ve SSB kültürleri hem de hemen hemen aynı idi. Bu gözlemler önceki yayında 3&#39;te sunulmuştur. glikoz düzeyleri her iki yöntem için kültür dönem...

Watch this Scientific Journal Video about Nutrient Regulation by Continuous Feeding for Large-scale Expansion of Mammalian Cells in Spheroids at JoVE.com

Nutrient Regulation by Continuous Feeding for Large-scale Expansion of Mammalian Cells in Spheroids

Nutrient regulation using continuous growth adjusted feeding improves growth rates of mammalian cell spheroids compared to intermittent batch feeding for cultures in stirred suspension bioreactors. This study demonstrates the methods required for establishing simple adjusted rate fed cultures.

Küresellerde Memeli Hücrelerinin Büyük ölçekli Genişleme için sürekli Beslenme ile Besin Yönetmeliği

In this demonstration, spheroids formed from the &#946;-TC6 insulinoma cell line were cultured as a model of manufacturing a mammalian islet cell product ...

nutrient-regulation-continuous-feeding-for-large-scale-expansion

Research

JoVE Journal

Bioengineering

7.7K Görüntüleme.  University of Minnesota. Nutrient regulation using continuous growth adjusted feeding improves growth rates of mammalian cell spheroids compared to intermittent batch feeding for cultures in stirred suspension bioreactors. This study demonstrates the methods required for establishing simple adjusted rate fed cultures.

Video: Nutrient Regulation by Continuous Feeding for Large-scale Expansion of Mammalian Cells in Spheroids

High Resolution 3D Imaging of Ex-Vivo Biological Samples by Micro CT

Non-destructive volume visualization can be achieved only by tomographic techniques, of which the most efficient is the x-ray micro computerized tomography (&mu;CT).
High resolution &mu;CT is a very versatile yet accurate (1-2 microns of resolution) technique for 3D examination of ex-vivo biological samples1, 2. As opposed to electron tomography, the &mu;CT allows the examination of up to 4 cm thick samples. This technique requires only few hours of measurement as compared to weeks in histology. In addition, &mu;CT does not rely on 2D stereologic models, thus it may complement and in some cases can even replace histological methods3, 4, which are both time consuming and destructive. Sample conditioning and positioning in &mu;CT is straightforward and does not require high vacuum or low temperatures, which may adversely affect the structure. The sample is positioned and rotated 180&deg; or 360&deg;between a microfocused x-ray source and a detector, which includes a scintillator and an accurate CCD camera, For each angle a 2D image is taken, and then the entire volume is reconstructed using one of the different available algorithms5-7. The 3D resolution increases with the decrease of the rotation step. The present video protocol shows the main steps in preparation, immobilization and positioning of the sample followed by imaging at high resolution.

High Resolution 3D Imaging of Ex-Vivo Biological Samples by Micro CT

Non-destructive volume visualization can be achieved only by tomographic techniques, of which the most efficient is the x-ray micro computerized tomography ( CT).

Yüksek Çözünürlüklü 3D Görüntüleme Ex-Vivo Biyolojik örnekler Micro CT

Non-destructive volume visualization can be achieved only by tomographic techniques, of which the most efficient is the x-ray micro computerized ...

Biyomühendislik

Continuous High-resolution Microscopic Observation of Replicative Aging in Budding Yeast

We demonstrate the use of a simple microfluidic setup, in which single budding yeast cells can be tracked throughout their entire lifespan. The microfluidic chip exploits the size difference between mother and daughter cells using an array of micropads. Upon loading, cells are trapped underneath these micropads, because the distance between the micropad and cover glass is similar to the diameter of a yeast cell (3-4 &mu;m). After the loading procedure, culture medium is continuously flushed through the chip, which not only creates a constant and defined environment throughout the entire experiment, but also flushes out the emerging daughter cells, which are not retained underneath the pads due to their smaller size. The setup retains mother cells so efficiently that in a single experiment up to 50 individual cells can be monitored in a fully automated manner for 5 days or, if necessary, longer. In addition, the excellent optical properties of the chip allow high-resolution imaging of cells during the entire aging process.

We describe here the operation of a microfluidic device that allows continuous and high-resolution microscopic imaging of single budding yeast cells during their complete replicative and/or chronological lifespan.

Replikatif sürekli yüksek çözünürlüklü Mikroskobik Gözlem geliştirici Maya Yaşlanma

We demonstrate the use of a simple microfluidic setup, in which single budding yeast cells can be tracked throughout their entire lifespan. The ...

Surgical Retrieval, Isolation and In vitro Expansion of Human Anterior Cruciate Ligament-derived Cells for Tissue Engineering Applications

Injury to the ACL is a commonly encountered problem in active individuals. Even partial tears of this intra-articular knee ligament lead to biomechanical deficiencies that impair function and stability. Current options for the treatment of partial ACL tears range from nonoperative, conservative management to multiple surgical options, such as: thermal modification, single-bundle repair, complete reconstruction, and reconstruction of the damaged portion of the native ligament. Few studies, if any, have demonstrated any single method for management to be consistently superior, and in many cases patients continue to demonstrate persistent instability and other comorbidities.
The goal of this study is to identify a potential cell source for utilization in the development of a tissue engineered patch that could be implemented in the repair of a partially torn ACL. A novel protocol was developed for the expansion of cells derived from patients undergoing ACL reconstruction. To isolate the cells, minced hACL tissue obtained during ACL reconstruction was digested in a Collagenase solution. Expansion was performed using DMEM/F12 medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (P/S). The cells were then stored at -80 &#186;C or in liquid nitrogen in a freezing medium consisting of DMSO, FBS and the expansion medium. After thawing, the hACL derived cells were then seeded onto a tissue engineered scaffold, PLAGA (Poly lactic-co-glycolic acid) and control Tissue culture polystyrene (TCPS). After 7 days, SEM was performed to compare cellular adhesion to the PLAGA versus the control TCPS. Cellular morphology was evaluated using immunofluorescence staining. SEM (Scanning Electron Microscope) micrographs demonstrated that cells grew and adhered on both PLAGA and TCPS surfaces and were confluent over the entire surfaces by day 7. Immunofluorescence staining showed normal, non-stressed morphological patterns on both surfaces. This technique is promising for applications in ACL regeneration and reconstruction.

Surgical Retrieval, Isolation and In vitro Expansion of Human Anterior Cruciate Ligament-derived Cells for Tissue Engineering Applications

For future applications as a patch to repair partial tears of the Anterior Cruciate Ligament (ACL), human ACL derived cells were isolated from tissue obtained during reconstructive procedures, expanded in vitro and grown on tissue engineered scaffolds. Cellular adhesion and morphology was then performed to confirm biocompatibility on scaffold surface.

Cerrahi Alma, İzolasyon ve<em&gt; In vitro</em&gt; Doku Mühendisliği Uygulamaları için İnsan Ön Çapraz Bağ-türevli Hücreleri Genişleme

Injury to the ACL is a commonly encountered problem in active individuals. Even partial tears of this intra-articular knee ligament lead to biomechanical ...

Luminescence Resonance Energy Transfer to Study Conformational Changes in Membrane Proteins Expressed in Mammalian Cells

Luminescence Resonance Energy Transfer, or LRET, is a powerful technique used to measure distances between two sites in proteins within the distance range of 10-100 &#197;. By measuring the distances under various ligated conditions, conformational changes of the protein can be easily assessed. With LRET, a lanthanide, most often chelated terbium, is used as the donor fluorophore, affording advantages such as a longer donor-only emission lifetime, the flexibility to use multiple acceptor fluorophores, and the opportunity to detect sensitized acceptor emission as an easy way to measure energy transfer without the risk of also detecting donor-only signal. Here, we describe a method to use LRET on membrane proteins expressed and assayed on the surface of intact mammalian cells. We introduce a protease cleavage site between the LRET fluorophore pair. After obtaining the original LRET signal, cleavage at that site removes the specific LRET signal from the protein of interest allowing us to quantitatively subtract the background signal that remains after cleavage. This method allows for more physiologically relevant measurements to be made without the need for purification of protein.

We describe here an improved Luminescence Resonance Energy Transfer (LRET) method where we introduce a protease cleavage site between the donor and acceptor fluorophore sites. This modification allows us to obtain specific LRET signals arising from membrane proteins of interest, allowing for the study of membrane proteins without protein purification.

Lüminesans Rezonans Enerji memeli hücrelerinde ifade Membran Proteinleri Konformasyonel Değişiklikleri Eğitim transfer

Luminescence Resonance Energy Transfer, or LRET, is a powerful technique used to measure distances between two sites in proteins within the distance range ...

Cultivation of Mammalian Cells Using a Single-use Pneumatic Bioreactor System

Recent advances in mammalian, insect, and stem cell cultivation and scale-up have created tremendous opportunities for new therapeutics and personalized medicine innovations. However, translating these advances into therapeutic applications will require in vitro systems that allow for robust, flexible, and cost effective bioreactor systems. There are several bioreactor systems currently utilized in research and commercial settings; however, many of these systems are not optimal for establishing, expanding, and monitoring the growth of different cell types. The culture parameters most challenging to control in these systems include, minimizing hydrodynamic shear, preventing nutrient gradient formation, establishing uniform culture medium aeration, preventing microbial contamination, and monitoring and adjusting culture conditions in real-time. Using a pneumatic single-use bioreactor system, we demonstrate the assembly and operation of this novel bioreactor for mammalian cells grown on micro-carriers. This bioreactor system eliminates many of the challenges associated with currently available systems by minimizing hydrodynamic shear and nutrient gradient formation, and allowing for uniform culture medium aeration. Moreover, the bioreactor&#8217;s software allows for remote real-time monitoring and adjusting of the bioreactor run parameters. This bioreactor system also has tremendous potential for scale-up of adherent and suspension mammalian cells for production of a variety therapeutic proteins, monoclonal antibodies, stem cells, biosimilars, and vaccines.

Using a pneumatic bioreactor, we demonstrate the assembly, operation, and performance of this single-use bioreactor system for the growth of mammalian cells.

Tek kullanımlık Pnömatik Biyoreaktör Sistemi kullanılarak memeli hücrelerinde yetiştirme

Recent advances in mammalian, insect, and stem cell cultivation and scale-up have created tremendous opportunities for new therapeutics and personalized ...

Cloning and Large-Scale Production of High-Capacity Adenoviral Vectors Based on the Human Adenovirus Type 5

High-capacity adenoviral vectors (HCAdV) devoid of all viral coding sequences represent one of the most advanced gene delivery vectors due to their high packaging capacity (up to 35 kb), low immunogenicity, and low toxicity. However, for many laboratories the use of HCAdV is hampered by the complicated procedure for vector genome construction and virus production. Here, a detailed protocol for efficient cloning and production of HCAdV based on the plasmid pAdFTC containing the HCAdV genome is described. The construction of HCAdV genomes is based on a cloning vector system utilizing homing endonucleases (I-CeuI and PI-SceI). Any gene of interest of up to 14 kb can be subcloned into the shuttle vector pHM5, which contains a multiple cloning site flanked by I-CeuI and PI-SceI. After I-CeuI and PI-SceI-mediated release of the transgene from the shuttle vector the transgene can be inserted into the HCAdV cloning vector pAdFTC. Because of the large size of the pAdFTC plasmid and the long recognition sites of the used enzymes associated with strong DNA binding, careful handling of the cloning fragments is needed. For virus production, the HCAdV genome is released by NotI digest and transfected into a HEK293 based producer cell line stably expressing Cre recombinase. To provide all adenoviral genes for adenovirus amplification, co-infection with a helper virus containing a packing signal flanked by loxP sites is required. Pre-amplification of the vector is performed in producer cells grown on surfaces and large-scale amplification of the vector is conducted in spinner flasks with producer cells grown in suspension. For virus purification, two ultracentrifugation steps based on cesium chloride gradients are performed followed by dialysis. Here tips, tricks, and shortcuts developed over the past years working with this HCAdV vector system are presented.

A protocol for generation of high-capacity adenoviral vectors lacking all viral coding sequences is presented. Cloning of transgenes contained in the vector genome is based on homing endonucleases. Virus amplification in producer cells grown as adherent cells and in suspension relies on a helper virus providing viral genes in trans.

İnsan Adenovirüs Tip 5 dayanarak Yüksek Kapasiteli Adenoviral Vektörler klonlanması ve Büyük Ölçekli Üretim

High-capacity adenoviral vectors (HCAdV) devoid of all viral coding sequences represent one of the most advanced gene delivery vectors due to their high ...

Culturing Mammalian Cells in Three-dimensional Peptide Scaffolds

A useful technique for culturing cells in a self-assembling nanofiber three-dimensional (3D) scaffold is described. This culture system recreates an environment that closely mimics the structural features of non-polarized tissue. Furthermore, the particular intrinsic nanofiber structure of the scaffold makes it transparent to visual light, which allows for easy visualization of the sample under microscopy. This advantage was largely used to study cell migration, organization, proliferation, and differentiation and thus any development of their particular cellular function by staining with specific dyes or probes. Furthermore, in this work, we describe the good performance of this system to easily study the redifferentiation of expanded human articular chondrocytes into cartilaginous tissue. Cells were encapsulated into self-assembling peptide scaffolds and cultured under specific conditions to promote chondrogenesis. Three-dimensional cultures showed good viability during the 4 weeks of the experiment. As expected, samples cultured with chondrogenic inducers (compared to non-induced controls) stained strongly positive for toluidine blue (which stains glycosaminoglycans (GAGs) that are highly present in cartilage extracellular matrix) and expressed specific molecular markers, including collagen type I, II and X, according to Western Blot analysis. This protocol is easy to perform and can be used at research laboratories, industries and for educational purposes in laboratory courses.

Here, we present a protocol to obtain 3D-culture systems in self-assembling peptide scaffolds to promote the differentiation of dedifferentiated human articular chondrocytes into cartilage-like tissue.

Üç boyutlu peptid iskele memeli hücrelerinde kültür

A useful technique for culturing cells in a self-assembling nanofiber three-dimensional (3D) scaffold is described. This culture system recreates an ...

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate (LAL) Assays

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause inflammation, fever, hypo- or hypertension, and, in extreme cases, can lead to tissue and organ damage that may become fatal. The amounts of endotoxin in pharmaceutical products, therefore, are strictly regulated. Among the methods available for endotoxin detection and quantification, the Limulus Amoebocyte Lysate (LAL) assay is commonly used worldwide. While any pharmaceutical product can interfere with the LAL assay, nano-formulations represent a particular challenge due to their complexity. The purpose of this paper is to provide a practical guide to researchers inexperienced in estimating endotoxins in engineered nanomaterials and nanoparticle-formulated drugs. Herein, practical recommendations for performing three LAL formats including turbidity, chromogenic and gel-clot assays are discussed. These assays can be used to determine endotoxin contamination in nanotechnology-based drug products, vaccines, and adjuvants.

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate LAL Assays

Detection of endotoxins in engineered nanomaterials represents one of the grand challenges in the field of nanomedicine. Here, we present a case study that describes the framework composed of three different LAL formats to estimate potential endotoxin contamination in nanoparticles.

Nano-formülasyonları endotoksin tespiti Limulus Amoebocyte Lysate (LAL) deneyleri kullanarak

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause ...

Cultivating a Three-dimensional Reconstructed Human Epidermis at a Large Scale

A three-dimensional human epidermis model reconstructed from neonatal primary keratinocytes is presented. Herein, a protocol for the cultivation process and the characterization of the model is described. Neonatal primary keratinocytes are grown submerged on permeable polycarbonate inserts and lifted to the air-liquid interface three days after seeding. After fourteen days of stimulation with defined growth factors and ascorbic acid in high calcium culture medium, the model is fully differentiated. Histological analysis revealed a completely stratified epidermis, mimicking the morphology of native human skin. To characterize the model and its barrier functions, protein levels and localization specific for early-stage keratinocyte differentiation (i.e., keratin 10), late-stage differentiation (i.e., involucrin, loricrin, and filaggrin) and tissue adhesion (i.e., desmoglein 1), were assessed by immunofluorescence. The tissue barrier integrity was further evaluated by measuring transepithelial electrical resistance. Reconstructed human epidermis was responsive to proinflammatory stimuli (i.e., lipopolysaccharide and tumor necrosis factor alpha), leading to increased cytokine release (i.e., interleukin 1 alpha and interleukin 8). This protocol represents a straightforward and reproducible in vitro method to cultivate reconstructed human epidermis as a tool to assess environmental effects and a broad range of skin-related studies.

This protocol describes a straightforward method to cultivate three-dimensional reconstituted human epidermis in a reproducible and robust manner. Additionally, it characterizes the structure-function relationship of the epidermal barrier model. The biological responses of the reconstituted human epidermis upon proinflammatory stimuli are also presented.

Büyük Ölçekte Üç Boyutlu Yeniden Yapılandırılmış İnsan Epidermisi Yetiştirmek

A three-dimensional human epidermis model reconstructed from neonatal primary keratinocytes is presented. Herein, a protocol for the cultivation process ...

Reliably Engineering and Controlling Stable Optogenetic Gene Circuits in Mammalian Cells

Reliable gene expression control in mammalian cells requires tools with high fold change, low noise, and determined input-to-output transfer functions, regardless of the method used. Toward this goal, optogenetic gene expression systems have gained much attention over the past decade for spatiotemporal control of protein levels in mammalian cells. However, most existing circuits controlling light-induced gene expression vary in architecture, are expressed from plasmids, and utilize variable optogenetic equipment, creating a need to explore characterization and standardization of optogenetic components in stable cell lines. Here, the study provides an experimental pipeline of reliable gene circuit construction, integration, and characterization for controlling light-inducible gene expression in mammalian cells, using a negative feedback optogenetic circuit as a case example. The protocols also illustrate how standardizing optogenetic equipment and light regimes can reliably reveal gene circuit features such as gene expression noise and protein expression magnitude. Lastly, this paper may be of use for laboratories unfamiliar with optogenetics who wish to adopt such technology. The pipeline described here should apply for other optogenetic circuits in mammalian cells, allowing for more reliable, detailed characterization and control of gene expression at the transcriptional, proteomic, and ultimately phenotypic level in mammalian cells.

Reliably controlling light-responsive mammalian cells requires the standardization of optogenetic methods. Toward this goal, this study outlines a pipeline of gene circuit construction, cell engineering, optogenetic equipment operation, and verification assays to standardize the study of light-induced gene expression using a negative-feedback optogenetic gene circuit as a case study.

Memeli Hücrelerinde Kararlı Optogenetik Gen Devrelerinin Güvenilir Mühendisliği ve Kontrolü

Reliable gene expression control in mammalian cells requires tools with high fold change, low noise, and determined input-to-output transfer functions, ...