이 작품은 각각 NSF 루이 보조금 MCB-0950372와 DMR-1105310받을 권리를 가진다 &quot;블랙 위도우 거미의 실크와 거미의 실크 접착제의 기계적 동작의 분자 특성화&quot;에 의해 지원되었다.

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관심의 어떠한 충돌 선언 없습니다.

이 방법론에서 냈지 합성 섬유는 천연 섬유에 비해 크기의 순서에 기계식입니다. 스풀 링 및 게시물 스핀 추첨 프로세스를 mechanizing하여 인간의 오류의 양을 줄임으로써, 샘플 간의 실험 유사 콘텐츠를 더 많은 제어와 획기적으로 줄일 수 있습니다. 우리의 방법론은 거미 유전자 가족의 다른 구성원의 cDNAs에서 인코딩 재조합 단백질에서 돌다가있는 다른 섬유의 기계적 특성?...

거미 실크는 고장력 강판, 방탄과 나일론을 포함한 밖으로 몇 가지 인공 물질을 수행 놀라운 기계적 성질을 가지고 있습니다. 1 거미들은 다양한 기계적 특성을 표시하는 적어도 6-7 가지 섬유 종류, 인장 강도와 확장성의 다양한 양의 설계된 각 스핀 특정 생물 학적 작업을 수행합니다.이 연구 과학자들은 급속하게 때문에 식인의 3,4. 때문에 그들의 뛰어난 기계적 특성, 그들의 biocompatibility, 그들의 무독성과 녹색 - 재료 자연의 차세대 biomaterials 같은 거미 실크 옷의 사용을 추구하고있다 그의 거미들도 악의에 찬 성격은 농업을 통해 거미 실크 옷을 수확하는 것은 산업 규모의 제조에 필요한 요구 사항을 충족하기위한 실용적인 전략이 아닙니다. 따라서 과학자들은로부터 합성 섬유의 방적 체외에서 결합 유전자 변형 생물체에 재조합 실크 단백질의 생산으로 향했다했습니다SE 정화 단백질이 있습니다. 전체 길이 재조합 거미 실크 단백질의 5-8 표현들은 고도로 반복적인 자연과 물리적 길이를 (&gt; 15킬로바이트) 등이 자신의 유전자 시퀀스의 본질적인 속성, GC 풍부한 컨텐츠 주어진 기술적으로 곤란했던과 편견되었습니다 알라닌과 글리신 코돈 사용. 날짜 9-11, 대부분의 실험실 부분 cDNA 시퀀스 또는 합성 유전자를 사용하여 주요 ampullate 실크 단백질 MaSp1 또는 MaSp2의 잘린 형태의 표현에 주력했습니다. 12-15 스피닝 합성 거미 실크 옷이 필요 도전적인 과정이다 여러 과학 분야, 그리고 방적 공정의 복잡한에 숙달과 지식 완전히 영상 표현하여 일반 대중에게 공개되지 않았습니다. 사실, 전세계 실험실만이는 거미 실크 cDNAs을 표현 실크 단백질 정화, 합성 섬유를 돌려서 후 스핀 추첨을 수행할 수있는 전문 지식을 가지고, 그리고 마지막으로 그들의 biomaterial 속성을 테스트합니다. 8,방적 합성 섬유에 대한 16,17 다른 접근 방식은 습식 및 건식 방사뿐만 아니라 electrospinning 방법을 포함하고있다 16,18,19 모든 절차는 공통점이 한 가지 목표가 -. 기계적 성질과 합성 거미 실크를 생산 프로토콜의 개발을 그 라이벌 천연 스레드 대규모 상업 생산 공정입니다. 여기 젖어도 회전하는 방법을 사용하여 실험실 규모의 인공 거미 실크 옷을 생성하는 절차를 설명합니다. 다른 방적 방법에 대한 상대, 젖은 지어내는 섬유 분석을위한 가장 일관성있는 결과를 생산하고있다. 우리는 그들의 정화이어서 박테리아 재조합 실크 단백질의 표현으로이 절차를 시작하는을 요약한 다음으로 스레드를 얻을 &quot;로-잣고&quot;섬유에 적용된 이후의 스핀 추첨 방법론을 포함하여, 방적 용 단백질 준비 단계를 설명 자연 거미 실크 옷의 품질을 접근 재료의 특성. 우리 methodologY가 밀접 실크 섬유의 자연적인 회전 과정을 모방하도록 설계하고 건축과 더 나아가 구를 및 COB-직조 거미. 20-22에서 비단 생산 땀샘의 기능은 우리의 전문 기술에 따라 크게 그리는되고, 우리는 필요로 결론 수사관은 궁극적인 강도, 궁극적인 변형, 그리고 섬유의 인성을 계산할 수 응력 - 변형 곡선을, 모략하는 tensometer를 사용하여 합성 섬유의 재료 특성을 결정하는 단계. 마지막으로,하지만 중요한 가치 때문에, 스풀 링 방적 및 그리기 apparatuses 오히려 정교하고 값비싼 맞춤형 장비를 구입하는 것보다 상용 부품을 사용하여 가정에서 만들 수 있습니다.

<table border="1"><tbody><tr><td> 시약 / 장비 </td><td> 회사 </td><td> 카탈로그 번호 </td><td> 댓글 </td></tr><tr><td> pBAD / TOPO ThioFusion 식 키트 </td><td> Invitrogen </td><td> K370-01 </td><td></td></tr><tr><td> 10X FastBreak 세포 용해 시약, </td><td> Promega </td><td> V857C </td><td></td></tr><tr><td> 니켈-NTA 아가로 오스 </td><td> Qiagen </td><td> 30,210 </td><td> 버퍼에 대한 지침이 포함되어 있습니다 </td></tr><tr><td> ProteoSilver 실버 얼룩 키트 </td><td> 시그마 - 올드 리치 </td><td> PROTSIL1-1KT </td><td></td></tr><tr><td> FreeZone Lyophilizer </td><td> Labconco </td><td> 7,960,041 </td><td> FreeZone 12Plus </td></tr><tr><td> Hexafluoroisopropanol (HFIP) </td><td> 시그마 - 올드 리치 </td><td> 52,512 </td><td></td></tr><tr><td> 주사기 </TD&gt; <td> 해밀턴 </td><td> 7657-01 </td><td> 250 μL </td></tr><tr><td> 바늘 </td><td> 해밀턴 </td><td> 7780-01 </td><td> 26s 게이지, 무딘 끝 이동식 바늘 </td></tr><tr><td> 주사기 펌프 </td><td> 하버드 장치 </td><td> 702,208 </td><td> 11Plus </td></tr><tr><td> 디지털 캘리퍼스 </td><td> Carrera </td><td> CP5906 </td><td> 0-150밀리미터 범위 </td></tr><tr><td> 스테인레스 스틸 포셉 </td><td> 세계 정밀 계측기 </td><td> 501,764 </td><td> 미니 뒤몽 # M5S </td></tr><tr><td> 모터 </td><td> 자연 밀 </td><td> 7,090,529 </td><td> 12VDC, 2 RPM 속도 </td></tr><tr><td> 리니어 액츄에이터 </td><td> 워너 전기 </td><td> 01-D024-0050-A06-LP-IP65 </td><td> 24VDC, 6 인치 범위 </td></tr><tr><td> 현미경 해부 </td><td> Leica 마이크로 시스템즈 </td><td> Leica MZ16 </td><td></td></tr><tr><td> 디지털 microscope 카메라 </td><td> Leica 마이크로 시스템즈 </td><td> DFC320 </td><td> 소프트웨어 : Leica 애플 리케이션 스위트 v2.8.1 </td></tr><tr><td> Vannas 가위 </td><td> 세계 정밀 계측기 </td><td> 500,260 </td><td></td></tr><tr><td> Microtensometer </td><td> 오로라 과학 </td><td> 310C </td><td> 5N 듀얼 모드 시스템 </td></tr></tbody></table>

synthetic spider silk production on a laboratory scale

사회가 진행하고 자원 scarcer되기 때문에, 새로운 기술을 육성하는 것이 점차 중요 해지고 고성능 특성을 가진 엔지니어 차세대 biomaterials니다. 이러한 새로운 구조 재료의 개발 비용 효율, 빠른이어야하며 처리 방법과 환경 친화적이고 지속 가능한 제품을 포함해야합니다. 거미는 biomimicry 그 라이벌 최고의 인조 및 천연 재료를위한 차세대 엔지니어링 재료의 풍부한 소스를 제공하고, 다양한 기계적 특성과 서로 다른 파이버 유형의 수많은 스핀. 자연 거미 실크의 대량의 컬렉션 허무이기 때문에, 합성 실크 생산이 스레드의 무제한 공급에 대한 액세스와 과학자를 제공할 수있는 능력을 가지고 있습니다. 스피닝 프로세스가 간편하고 완벽하게 될 수 있습니다 따라서, 인공 거미 섬유 갑옷에서부터 애플 리케이션의 광범위한, 수술 봉합에 대한 잠재적인 사용을초, 로프 및 케이블, 타이어, 악기를위한 문자열, 그리고 항공 및 우주 항공 기술에 대한 복합 재료. 합성 실크 생산 과정을 진행하고 회전하는 스핀에서 그들의 물질적 속성에서 낮은 편차를 표시할 수 섬유를 양보하기 위해, 우리는 박테리아, 정화 및 단백질의 농도에서 재조합 거미 실크 단백질의 표현을 통합 젖어도 회전하는 프로토콜을 개발 , 섬유 압출 및 기계적 이후 스핀 치료로 따라갔다. 이것은 실험실 규모의 인조 실크 섬유를 돌려서 분석 단계별 프로세스를 보여 최초의 시각적 표현이다. 또한 동일한 회전 마약에서 냈지 섬유 간의 다양성의 도입을 최소화하기 위해 세부 사항을 제공합니다. 이하 이러한 방법은 자연 거미 실크 옷을 능가하는 높은 품질의 섬유로 이어지는, 인조 실크 생산의 공정을 추진합니다.

Watch this Scientific Journal Video about Synthetic Spider Silk Production on a Laboratory Scale at JoVE.com

Synthetic Spider Silk Production on a Laboratory Scale

거미 실크 옷의 뛰어난 기계적 및 생화 학적 특성에도 불구하고,이 재료는 종래의 방법으로 대량 수확 할 수 없습니다. 여기 차세대 biomaterials 같은 거미 실크의 생산과 사용을 공부 수사관을위한 중요한 과정입니다 인공 거미 실크 섬유를 스핀하는 효율적인 전략을 설명합니다.

실험실 규모의 합성 거미의 실크 생산

As society progresses and resources become scarcer, it is becoming increasingly important to cultivate new technologies that engineer next generation ...

synthetic-spider-silk-production-on-a-laboratory-scale

Research

JoVE Journal

Bioengineering

26.5K 조회수.  University of the Pacific. 거미 실크 옷의 뛰어난 기계적 및 생화 학적 특성에도 불구하고,이 재료는 종래의 방법으로 대량 수확 할 수 없습니다. 여기 차세대 biomaterials 같은 거미 실크의 생산과 사용을 공부 수사관을위한 중요한 과정입니다 인공 거미 실크 섬유를 스핀하는 효율적인 전략을 설명합니다.

비디오: Synthetic Spider Silk Production on a Laboratory Scale

Air Filter Devices Including Nonwoven Meshes of Electrospun Recombinant Spider Silk Proteins

Based on the natural sequence of Araneus diadematus Fibroin 4 (ADF4), the recombinant spider silk protein eADF4(C16) has been engineered. This highly repetitive protein has a molecular weight of 48kDa and is soluble in different solvents (hexafluoroisopropanol (HFIP), formic acid and aqueous buffers). eADF4(C16) provides a high potential for various technical applications when processed into morphologies such as films, capsules, particles, hydrogels, coatings, fibers and nonwoven meshes. Due to their chemical stability and controlled morphology, the latter can be used to improve filter materials. In this protocol, we present a procedure to enhance the efficiency of different air filter devices, by deposition of nonwoven meshes of electrospun recombinant spider silk proteins. Electrospinning of eADF4(C16) dissolved in HFIP results in smooth fibers. Variation of the protein concentration (5-25% w/v) results in different fiber diameters (80-1,100 nm) and thus pore sizes of the nonwoven mesh.
Post-treatment of eADF4(C16) electrospun from HFIP is necessary since the protein displays a predominantly &alpha;-helical secondary structure in freshly spun fibers, and therefore the fibers are water soluble. Subsequent treatment with ethanol vapor induces formation of water resistant, stable &beta;-sheet structures, preserving the morphology of the silk fibers and meshes. Secondary structure analysis was performed using Fourier transform infrared spectroscopy (FTIR) and subsequent Fourier self-deconvolution (FSD).
The primary goal was to improve the filter efficiency of existing filter substrates by adding silk nonwoven layers on top. To evaluate the influence of electrospinning duration and thus nonwoven layer thickness on the filter efficiency, we performed air permeability tests in combination with particle deposition measurements. The experiments were carried out according to standard protocols.

Spider silk fibers display extraordinary mechanical properties. Engineered Araneus diadematus Fibroin 4 (eADF4) can be processed into nonwoven meshes using electrospinning. Here, the eADF4 nonwoven meshes are used to improve the performance of air filtering devices.

전기 방사 재조합 거미 실크 단백질의 부직포 메쉬를 포함하여 공기 정화 장치

Based on the natural sequence of Araneus diadematus Fibroin 4 (ADF4), the recombinant spider silk protein eADF4(C16) has been engineered. This highly ...

연구

생체공학

Designing Silk-silk Protein Alloy Materials for Biomedical Applications

Fibrous proteins display different sequences and structures that have been used for various applications in biomedical fields such as biosensors, nanomedicine, tissue regeneration, and drug delivery. Designing materials based on the molecular-scale interactions between these proteins will help generate new multifunctional protein alloy biomaterials with tunable properties. Such alloy material systems also provide advantages in comparison to traditional synthetic polymers due to the materials biodegradability, biocompatibility, and tenability in the body. This article used the protein blends of wild tussah silk (Antheraea pernyi) and domestic mulberry silk (Bombyx mori) as an example to provide useful protocols regarding these topics, including how to predict protein-protein interactions by computational methods, how to produce protein alloy solutions, how to verify alloy systems by thermal analysis, and how to fabricate variable alloy materials including optical materials with diffraction gratings, electric materials with circuits coatings, and pharmaceutical materials for drug release and delivery. These methods can provide important information for designing the next generation multifunctional biomaterials based on different protein alloys.

Blending is an efficient approach to generate biomaterials with a broad range of properties and combined features. By predicting the molecular interactions between different natural silk proteins, new silk-silk protein alloy platforms with tunable mechanical resiliency, electrical response, optical transparency, chemical processability, biodegradability, or thermal stability can be designed.

바이오 메디컬 응용 프로그램에 대한 실크 실크 단백질 합금 재료 설계

Fibrous proteins display different sequences and structures that have been used for various applications in biomedical fields such as biosensors, ...

Production and Targeting of Monovalent Quantum Dots

The multivalent nature of commercial quantum dots (QDs) and the difficulties associated with producing monovalent dots have limited their applications in biology, where clustering and the spatial organization of biomolecules is often the object of study. We describe here a protocol to produce monovalent quantum dots (mQDs) that can be accomplished in most biological research laboratories via a simple mixing of CdSe/ZnS core/shell QDs with phosphorothioate DNA (ptDNA) of defined length. After a single ptDNA strand has wrapped the QD, additional strands are excluded from the surface. Production of mQDs in this manner can be accomplished at small and large scale, with commercial reagents, and in minimal steps. These mQDs can be specifically directed to biological targets by hybridization to a complementary single stranded targeting DNA. We demonstrate the use of these mQDs as imaging probes by labeling SNAP-tagged Notch receptors on live mammalian cells, targeted by mQDs bearing a benzylguanine moiety.

We provide detailed instructions for the preparation of monovalent targeted quantum dots (mQDs) from phosphorothioate DNA of defined length. DNA wrapping occurs in high yield, and therefore, products do not require purification. We demonstrate the use of the SNAP tag to target mQDs to cell-surface receptors for live-cell imaging applications.

생산 및 일가 양자점 타겟팅

The multivalent nature of commercial quantum dots (QDs) and the difficulties associated with producing monovalent dots have limited their applications in ...

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.

인간 아데노 바이러스 유형 5를 기반으로 대용량 아데노 바이러스 벡터의 클로닝 및 대규모 생산

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

Engineered 3D Silk-collagen-based Model of Polarized Neural Tissue

Despite huge efforts to decipher the anatomy, composition and function of the brain, it remains the least understood organ of the human body. To gain a deeper comprehension of the neural system scientists aim to simplistically reconstruct the tissue by assembling it in vitro from basic building blocks using a tissue engineering approach. Our group developed a tissue-engineered silk and collagen-based 3D brain-like model resembling the white and gray matter of the cortex. The model consists of silk porous sponge, which is pre-seeded with rat brain-derived neurons, immersed in soft collagen matrix. Polarized neuronal outgrowth and network formation is observed with separate axonal and cell body localization. This compartmental architecture allows for the unique development of niches mimicking native neural tissue, thus enabling research on neuronal network assembly, axonal guidance, cell-cell and cell-matrix interactions and electrical functions.

Insight into the complex actions of the brain requires advanced research tools. Here we demonstrate a novel silk-collagen-based 3D engineered model of neural tissue resembling brain-like architecture. The model can be used to study neuronal network assembly, axonal guidance, cell-cell interactions and electrical activity.

편광 신경 조직의 엔지니어링 3D 실크 콜라겐 기반 모델

Despite huge efforts to decipher the anatomy, composition and function of the brain, it remains the least understood organ of the human body. To gain a ...

Power Input Measurements in Stirred Bioreactors at Laboratory Scale

The power input in stirred bioreactors is an important scaling-up parameter and can be measured through the torque that acts on the impeller shaft during rotation. However, the experimental determination of the power input in small-scale vessels is still challenging due to relatively high friction losses inside typically used bushings, bearings and/or shaft seals and the accuracy of commercially available torque meters. Thus, only limited data for small-scale bioreactors, in particular single-use systems, is available in the literature, making comparisons among different single-use systems and their conventional counterparts difficult.
This manuscript provides a protocol on how to measure power inputs in benchtop scale bioreactors over a wide range of turbulence conditions, which can be described by the dimensionless Reynolds number (Re). The aforementioned friction losses are effectively reduced by the use of an air bearing. The procedure on how to set up, conduct and evaluate a torque-based power input measurement, with special focus on cell culture typical agitation conditions with low to moderate turbulence (100 &#60; Re &#60; 2&#183;104), is described in detail. The power input of several multi-use and single-use bioreactors is provided by the dimensionless power number (also called Newton number, P0), which is determined to be in the range of P0 &#8776; 0.3 and P0 &#8776; 4.5 for the maximum Reynolds numbers in the different bioreactors.

The power input in stirred bioreactors can be measured through the torque that acts on the impeller shaft during rotation. This manuscript describes how an air bearing can be used to effectively reduce friction losses observed in mechanical seals and improve the accuracy of power input measurements in small-scale vessels.

The power input in stirred bioreactors is an important scaling-up parameter and can be measured through the torque that acts on the impeller shaft during ...

Material Formation of Recombinant Spider Silks through Aqueous Solvation using Heat and Pressure

Many spiders produce seven types of silks. Six of the silks are fiber in form when produced by the spiders. These fibers are not water soluble. In order to reproduce the remarkable mechanical properties of spider silks, they must be produced in heterologous hosts as spiders are both territorial and cannibalistic. The synthetic analogs of spider silk also tend to be insoluble in aqueous solutions. Thus, a large percentage of research in recombinant spider silks rely upon organic solvents that are detrimental to large scale production of materials. Our group&#8217;s method forces the solvation of these recombinant spider silks into water. Remarkably, when these proteins are prepared using this method of heat and pressure, a wide range of material forms can be prepared from the same solution of recombinant spider silk proteins (rSSp) including: films, fibers, sponge, hydrogel, lyogel, and adhesives. This article demonstrates the production of the solvated rSSp and material forms in a manner that is more easily understood than from written materials and methods alone.

Here, we present a protocol to produce water soluble recombinant spider silk protein solutions and the material forms that can be formed from those solutions.

열과 압력을 이용한 수성 용 매 화를 통한 재조합 거미 실크의 재료 형성

Many spiders produce seven types of silks. Six of the silks are fiber in form when produced by the spiders. These fibers are not water soluble. In order ...

Large-Scale, Automated Production of Adipose-Derived Stem Cell Spheroids for 3D Bioprinting

Adipose-derived stromal/stem cells (ASCs) are a subpopulation of cells found in the stromal vascular fraction of human subcutaneous adipose tissue recognized as a classical source of mesenchymal stromal/stem cells. Many studies have been published with ASCs for scaffold-based tissue engineering approaches, which mainly explored the behavior of these cells after their seeding on bioactive scaffolds. However, scaffold-free approaches are emerging to engineer tissues in vitro and in vivo, mainly by using spheroids, to overcome the limitations of scaffold-based approaches.
Spheroids are 3D microtissues formed by the self-assembly process. They can better mimic the architecture and microenvironment of native tissues, mainly due to the magnification of cell-to-cell and cell-to-extracellular matrix interactions. Recently, spheroids are mainly being explored as disease models, drug screening studies, and building blocks for 3D bioprinting. However, for 3D bioprinting approaches, numerous spheroids, homogeneous in size and shape, are necessary to biofabricate complex tissue and organ models. In addition, when spheroids are produced automatically, there is little chance for microbiological contamination, increasing the reproducibility of the method.
The large-scale production of spheroids is considered the first mandatory step for developing a biofabrication line, which continues in the 3D bioprinting process and finishes in the full maturation of the tissue construct in bioreactors. However, the number of studies that explored the large-scale ASC spheroid production are still scarce, together with the number of studies that used ASC spheroids as building blocks for 3D bioprinting. Therefore, this article aims to show the large-scale production of ASC spheroids using a non-adhesive micromolded hydrogel technique spreading ASC spheroids as building blocks for 3D bioprinting approaches.

Here, we describe the large-scale production of adipose-derived stromal/stem cell (ASC) spheroids using an automated pipetting system to seed the cell suspension, thus ensuring homogeneity of spheroid size and shape. These ASC spheroids can be used as building blocks for 3D bioprinting approaches.

3D 바이오프린팅을 위한 지방유래 줄기세포 구상체의 대규모, 자동화 생산

Adipose-derived stromal/stem cells (ASCs) are a subpopulation of cells found in the stromal vascular fraction of human subcutaneous adipose tissue ...

용해 가능한 마이크로캐리어를 기반으로 한 부착 세포의 폐쇄 시스템 생산

Large-Scale Cell Production Based on GMP-Grade Dissolvable Porous Microcarriers

Researchers in the cell and gene therapy (CGT) industry have long faced a formidable challenge in the efficient and large-scale expansion of cells. To address the primary shortcomings of the two-dimensional (2D) planar culturing system, we innovatively developed an automated closed industrial scale cell production (ACISCP) platform based on a GMP-grade, dissolvable, and porous microcarrier for the 3D culture of adherent cells, including human mesenchymal stem/stromal cells (hMSCs), HEK293T cells, and Vero cells. To achieve large-scale expansion, a two-stage expansion was conducted with 5 L and 15 L stirred-tank bioreactors to yield 1.1 x 1010 hMSCs with an overall 128-fold expansion within 9 days. The cells were harvested by completely dissolving the microcarriers, concentrated, washed and formulated with a continuous-flow centrifuge-based cell processing system, and then aliquoted with a cell filling system. Compared with 2D planar culture, there are no significant differences in the quality of hMSCs harvested from 3D culture. We have also applied these dissolvable porous microcarriers to other popular cell types in the CGT sector; specifically, HEK293T cells and Vero cells have been cultivated to peak cell densities of 1.68 x 107 cells/mL and 1.08 x 107 cells/mL, respectively. This study provides a protocol for using a bioprocess engineering platform harnessing the characteristics of GMP-grade dissolvable microcarriers and advanced closed equipment to achieve the industrial-scale manufacturing of adherent cells.

저자 스포트라이트: Advancing Cell Therapy Manufacturing with Dissolvable Microcarriers 

Here, we present a protocol to achieve the large-scale manufacturing of adherent cells through a fully closed system based on GMP-grade dissolvable microcarriers. The cultivation of human mesenchymal stem cells, HEK293T cells, and Vero cells was validated and met both quantity demands and quality criteria for the cell and gene therapy industry.

GMP 등급의 용해성 다공성 마이크로캐리어를 기반으로 한 대규모 셀 생산

Researchers in the cell and gene therapy (CGT) industry have long faced a formidable challenge in the efficient and large-scale expansion of cells. To ...