우리는 그들의 지원 및 기술 지원에 대한 공동 과학, NYUMC의 현미경 코어, 사무실에서 홍콩 연구실 박사 연 덩의 모든 회원에게 감사하고 싶습니다. 이 작품은 AIDS 연구 뉴욕 대학 센터 (CFAR, NIH 보조금 AI027742)에서 파일럿 보조금에 의해 지원되었다.

1. 플라스미드 DNA의 정제 <ol><li> DH5α E.coli 관할 세포에 플라스미드 DNA 진핵 세포 인코딩 EGFP (PCX-EGFP)를 변환 할 수 있습니다. 빈 PCX 벡터는 또한 대조군으로 사용하실 수 있습니다. </li><li> 문화와 수확 DH5α E.coli 세포를하고 Qiagen EndoFree 플라스미드 정화 핸드북에 따라 pDNA를 정화. </li><li> 필터 pDNA의 0.22 μm PVDF 살균 필터를 통해 솔루션 및 사용하기 전까지 -20 ° C에서 저장합니다. </li></ol> 2. 문신 시스템 준비 <ol><li> 제조업체의 지침에 따라 전원 공급 장치에 핸드 헬드 장치와 제어 페달을 연결합니다. </li><li> 약 100 Hz에서에 바늘 진동 주파수를 설정합니다. 우리가 (스텔스 로터리 문신 시스템) 선택한 문신 장치, 전원 공급 장치의 다이얼은 4 볼트로 설정해야합니다. 사용자 매뉴얼이나 기타 문신 시스템에 대한 기술 지원을 참조하십시오. </li><li> 바늘 AR를 검사사용하기 전에 선. 우리는 동물 당 하나의 바늘 어레이를 사용하는 것이 좋습니다. 바늘은 비눗물로 세척하고 autoclaved 후에 재사용 할 수 있습니다. </li><li> 핸드 헬드 장치에 바늘 어레이를 설치하고 느슨하게 플라스틱 손잡이를 첨부합니다. </li><li> 위 또는 아래 플라스틱 손잡이를 이동하여 적절한 설정으로 바늘 깊이를 조정합니다. 우리는 우리의 마우스 실험 약 0.5 mm의 바늘 깊이를 사용했습니다. </li><li> 바늘 깊이가 제대로 설정되어 플라스틱 손잡이를 조여하고, 문신 시스템은 사용할 수 있습니다. </li></ol> 3. 동물 면도 <ol><li> 문신 치료 사이트를 선택합니다. 이 사이트는 상대적으로 회사 평면, 그리고 살의 피부의 지역, 예를 들어, 동물의 hindleg의 측면해야합니다. </li><li> 동물의 체중에 따라 케타민 (90 MG / kg)과 xylazine (5 밀리그램 / kg)의 혼합물 balb / C 마우스를 마취. 다른 동물의 경우 교육 기관의 마취 지침을 참조하십시오. </li><li> 을 확인동물을 확인하기 위해 발을 곤란하게하여 동물의 반사 신경이 제대로 anesthetized 있습니다. </li><li> 천천히 그리고 조심스럽게 동물의 hindlegs에서 머리를 잘라하는 전기 트리머를 사용합니다. 계획 문신 사이트와 주변 지역에서 머리를 제거합니다. </li><li> 일회용 안전 면도기와 잔류 짧은 머리를 제거합니다. 동물의 피부를 잘라하지 않도록주의하십시오. 탈모 용 크림이 다른 헤어 제거 방법으로 사용할 수 있습니다, 그러나, 우리는 탈모 용 크림으로 면도를 선호. </li><li> 압축 공기 필요한 경우로 피부의 빗나간 컷 머리카락을 제거합니다. </li></ol> 4. 문신의 플라스미드 DNA의 전달 <ol><li> 예방 접종 프로토콜은 이전에 설정 한 경우, 복용하고 적용 할 수있는 DNA 솔루션의 볼륨은 그에 따라 계산해야합니다. 우리는 문신 과정을보다 손쉽게 관리 할 수​​ 실제와 같은 작은 볼륨으로 사용하는 것이 좋습니다. 이 EGFP 데모에서, 우리는 사기꾼의 DNA 솔루션의 7.5 μl를 사용약 1cm 2의 문신 지역에 적용되었습니다 0.25 밀리그램 / ML의 centration. 예방 접종 실험, 높은 pDNA 농도는 강력한 면역 반응의 1,2를 유도해야 할 수 있습니다. </li><li> 문신 사이트에 직접 pipetting하여 DNA 솔루션을 적용합니다. 또는 pipetting하여 플라스틱 그립에 DNA 솔루션을로드하기 때문에 액체는 바늘 및 플라스틱 그립의 끝 사이에 일시 중지됩니다. 데모의 동영상을 참조하십시오. 우리는 두 가지 방법 사이에 눈에 띄는 차이를 관찰 없습니다. 그러나, 플라스틱 그립에 솔루션을로드하는 것은 예를 들어, 수직 피부 표면에 바람직 할 수있다. </li><li> 바늘 진동을 시작하고 문신 사이트에있는 동물의 피부에 빛을 압력으로 바늘 어레이를 배치합니다. 그런 다음 전체 문신 영역에 플라스미드 DNA를 제공하기 위해 선형 방식으로 부드럽게 천천히 왕복 운동 바늘을 이동합니다. 바늘과 피하기 위해 피부 사이에 90도 각도를 유지피부에 열상. </li><li> 피부를 관찰. 마모와 염증은 정상입니다. 출혈이 발생하면, 문신 프로세스를 중지하고 바늘 깊이를 줄이십시오. </li><li> 약 1 분의 바늘 운동을 계속 한 다음 문신 프로세스를 중지합니다. </li><li> 문신 사이트에 국소 진통제의 얇은 층을 적용하기 위해 깨끗한 면봉을 사용합니다. 이러한 실버 Sulfadiazine 크림 (SSD 크림) 또는 Neosporin 연고와 같은 국소 진통제는 동물의 통증이나 고통을 완화 할 수 있습니다, 따라서 그것은 마취 떨어지기 전에 적용하는 것이 좋습니다. 통증이나 고통 (걸음 걸이, 비활성 변경)의 흔적을 다음 날 동물을 관찰합니다. 신호가 발생하면 진통제 크림을 다시 적용합니다. </li></ol> 5. 항원 표현의 확인 <ol><li> 문신 치료 (다른 pDNA 구조가 서로 다른 표현 시간을해야 할 수 있습니다) 후 48 시간이 마우스는 CO 2 마취법으로 euthanized 있습니다. 기타의 경우동물, 네 기관의 안락사 지침을 참조하십시오. </li><li> 문신 사이트에서 피부를 해부, 4 ° C 하룻밤에 4 % paraformaldehyde에 조직을 해결할 수 있습니다. </li><li> 70 % 에탄올로 조직을 헹구고, 영상에 1X PBS로 전송. </li><li> EGFP 신호를 확인하기 위해 공 촛점 현미경을 사용하여 전체 조직을 검사합니다. 또는 피부 조직은 파라핀 왁스에 포함하고 형광 현미경 섹션에서 검토 할 수 있습니다. </li></ol>

<ol>
	<li>Bins, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+and+potent+DNA+vaccination+strategy+defined+by+in+vivo+monitoring+of+antigen+expression.">A rapid and potent DNA vaccination strategy defined by in vivo monitoring of antigen expression.</a> Nat Med. 11, 899-904 (2005).</li><li>Pokorna, D., Rubio, I., Muller, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA-vaccination+via+tattooing+induces+stronger+humoral+and+cellular+immune+responses+than+intramuscular+delivery+supported+by+molecular+adjuvants.">DNA-vaccination via tattooing induces stronger humoral and cellular immune responses than intramuscular delivery supported by molecular adjuvants.</a> Genet. Vaccines Ther. 6, 4 (2008).</li><li>van den Berg, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+intradermal+vaccination+by+DNA+tattooing+in+human+skin.">Optimization of intradermal vaccination by DNA tattooing in human skin.</a> Hum. Gene Ther. 20, 181-189 (2009).</li><li>Liu, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+vaccines:+a+review.">DNA vaccines: a review.</a> J. Intern. Med. 253, 402-410 (2003).</li><li>Koide, Y., Nagata, T., Yoshida, A., Uchijima, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+vaccines.">DNA vaccines.</a> Jpn. J. Pharmacol. 83, 167-174 (2000).</li><li>Peachman, K. K., Rao, M., Alving, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunization+with+DNA+through+the+skin.">Immunization with DNA through the skin.</a> Methods. 31, 232-242 (2003).</li><li>Verstrepen, B. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+HIV-1+specific+T-cell+responses+by+short-interval+DNA+tattooing+as+compared+to+intramuscular+immunization+in+non-human+primates.">Improved HIV-1 specific T-cell responses by short-interval DNA tattooing as compared to intramuscular immunization in non-human primates.</a> Vaccine. 26, 3346-3351 (2008).</li><li>Potthoff, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunogenicity+and+efficacy+of+intradermal+tattoo+immunization+with+adenoviral+vector+vaccines.">Immunogenicity and efficacy of intradermal tattoo immunization with adenoviral vector vaccines.</a> Vaccine. 27, 2768-2774 (2009).</li><li>Quaak, S. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+tattoo+vaccination:+effect+on+plasmid+purity+and+transfection+efficiency+of+different+topoisoforms.">DNA tattoo vaccination: effect on plasmid purity and transfection efficiency of different topoisoforms.</a> J. Control Release. 139, 153-159 (2009).</li><li>Pokorna, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vaccination+with+human+papillomavirus+type+16-derived+peptides+using+a+tattoo+device.">Vaccination with human papillomavirus type 16-derived peptides using a tattoo device.</a> Vaccine. 27, 3519-3529 (2009).</li></ol>

관심 없음 충돌이 선언 없습니다.

이 조작을 필요로하거나, 라이브 또는 감소 병원체 4로 백신을 제공하지 않는 한 DNA 예방 접종은 기존의 예방 접종 전략보다 더 안전한 것으로 간주됩니다. 그러나, DNA 예방 접종의 결과는 배달 경로에 크게 의존한다. 피부는 Langerhans 세포와 수지상 세포 1, 따라서 immunogenicity 및 액세스 5,6의 용이성의 측면에서 예방 접종을위한 이상적인 사이트와 항원 제시 세포에 풍부하?...

<table border="1"><tbody><tr><td> 시약 / 재료의 이름 </td><td> 회사 </td><td> 카탈로그 번호 </td><td> 코멘트 </td></tr><tr><td> PCX-EGFP 플라스미드 DNA </td><td> Clontech </td><td></td><td></td></tr><tr><td> 스텔스 로터리 문신 시스템 </td><td> 전세계 문신 공급 </td><td> 은신-L </td><td></td></tr><tr><td> 문신 바늘 </td><td> 전세계 문신 공급 </td><td> 1207RSB </td><td></td></tr><tr><td> EndoFree 플라스미드 맥시 키트 </td><td> Qiagen </td><td> 12,362 </td><td></td></tr><tr><td> 0.22 μm PVDF 무균 필터 </td><td> Millipore </td><td> SLGV013SL </td><td></td></tr><tr><td> 전기 헤어 트리머 </td><td> 상업적으로 이용 가능한 </td><td></td><td></td></tr><tr><td> 일회용 안전 면도기 </td><td> 상업적으로 이용 가능한 </td><td></td><td></td></tr><tr><td> 은Sulfadiazine 크림 </td><td> 왓슨 </td><td> NDC 0591-0810-55 </td><td></td></tr><tr><td> 케타민 HCL </td><td></td><td> NDC 0856-2012-01 </td><td></td></tr><tr><td> Zylazine 무균 솔루션 </td><td></td><td> 나다 139-236 </td><td></td></tr></tbody></table>

skin tattooing as a novel approach for dna vaccine delivery

핵산 기반의 예방 접종은 성장 관심 주제 immunologically 중요한 항원을 인코딩 특히 플라스미드 DNA (pDNA)입니다. 엔지니어링 pDNA이 백신에 투여 한 후,이 베꼈와 면역 시스템의 반응을 유도 할 수 immunogen 단백질로 번역되어 있습니다. DNA 백신을 제공하는 대부분의 방법은 조사 된 있지만 각 배달 경로는 자신의 장점과 함정이 있습니다. 피부 문신은 안전 비용 효과적인지, 그리고 편리 소설 기법이다. 또한, 바늘 자국으로 구멍은 강력한 도움이되는 역할을 수 있습니다. 여기, 우리가) 플라스미드 DNA 인코딩의 intradermal 배달을 보여 강화 된 녹색 형광 공 촛점 현미경을 사용하여 피부 세포의 EGFP의 효과적인 표현을 확인 문신 장치 및 B)를 사용하여 마우스 모델에서 단백질 (PCX-EGFP).

509 나노 미터에서 488 나노 미터와 방출 피크에서 여기 피크와 EGFP의 표현은 마우스 피부 세포에서 관찰 할 수 있습니다. 약 3 × 포함 DNA의 1.875 μg 선량에서 10 플라스미드 17 사본, 우리는 일반적으로 2 영역을 문신 1cm에서 10-20 EGFP 신호를 관찰했다. transfected 세포의이 상대적으로 낮은 숫자는 이전 연구 (3)의 결과와 일치한다. EGFP 표현 (그림 1) 플라스미드 EGFP가 ...

Watch this Scientific Journal Video about Skin Tattooing As A Novel Approach For DNA Vaccine Delivery at JoVE.com

Skin Tattooing As A Novel Approach For DNA Vaccine Delivery

피부 문신은 intradermally 배달 DNA 백신에 대한 강력한하고 안전한 방법입니다. 여기 DNA 플라스미드 인코딩 EGFP는 실험실 쥐의 피부에 문신으로 전달되고 피부 세포에서 EGFP의 표현 그 다음 공 촛점 현미경으로 검사합니다.

피부는 DNA 백신 배달을위한 새로운 접근 방식으로 문신

Nucleic acid-based vaccination is a topic of growing interest, especially plasmid DNA (pDNA) encoding immunologically important antigens. After the ...

skin-tattooing-as-a-novel-approach-for-dna-vaccine-delivery

Research

JoVE Journal

Bioengineering

15.8K 조회수.  New York University School of Medicine. 피부 문신은 intradermally 배달 DNA 백신에 대한 강력한하고 안전한 방법입니다. 여기 DNA 플라스미드 인코딩 EGFP는 실험실 쥐의 피부에 문신으로 전달되고 피부 세포에서 EGFP의 표현 그 다음 공 촛점 현미경으로 검사합니다.

비디오: Skin Tattooing As A Novel Approach For DNA Vaccine Delivery

Cell Squeezing as a Robust, Microfluidic Intracellular Delivery Platform

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This technology has shown potential in addressing previously challenging applications; including, delivery to primary immune cells, cell reprogramming, carbon nanotube, and quantum dot delivery. This vector-free microfluidic platform relies on mechanical disruption of the cell membrane to facilitate cytosolic delivery of the target material. Herein, we describe the detailed method of use for these microfluidic devices including, device assembly, cell preparation, and system operation. This delivery approach requires a brief optimization of device type and operating conditions for previously unreported applications. The provided instructions are generalizable to most cell types and delivery materials as this system does not require specialized buffers or chemical modification/conjugation steps. This work also provides recommendations on how to improve device performance and trouble-shoot potential issues related to clogging, low delivery efficiencies, and cell viability.

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This protocol provides detailed steps on how to use the system for a broad range of applications.

강력한, 미세 유체 세포 내 전달 플랫폼으로 휴대 뒤쪽에서

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This ...

연구

생체공학

An Improved Method for the Preparation of Type I Collagen From Skin

Soluble type 1 collagen (COL1) is used extensively as an adhesive substrate for cell cultures and as a cellular scaffold for regenerative applications. Clinically, this protein is widely used for cosmetic surgery, dermal injections, bone grafting, and reconstructive surgery. The sources of COL1 for these procedures are commonly nonhuman, which increases the potential for inflammation and rejection as well as xenobiotic disease transmission. In view of this, a method to efficiently and quickly purify COL1 from limited quantities of autologously-derived tissues would circumvent many of these issues; however, standard isolation protocols are lengthy and often require large quantities of collagenous tissues. Here, we demonstrate an efficient COL1 extraction method that reduces the time needed to isolate and purify this protein from about 10 days to less than 3 hr. We chose the dermis as our tissue source because of its availability during many surgical procedures. This method uses traditional extraction buffers combined with forceful agitation and centrifugal filtration to obtain highly-pure, soluble COL1 from small amounts of corium. Briefly, dermal biopsies are washed thoroughly in ice-cold dH2O after removing fat, connective tissue, and hair. The skin samples are stripped of noncollagenous proteins and polysaccharides using 0.5 M sodium acetate and a high speed bench-top homogenizer. Collagen from residual solids is subsequently extracted with a 0.075 M sodium citrate buffer using the homogenizer. These extracts are purified using 100,000 MW cut-off centrifugal filters that yield COL1 preparations of comparable or superior quality to commercial products or those obtained using traditional procedures. We anticipate this method will facilitate the utilization of autologously-derived COL1 for a multitude of research and clinical applications.

Traditional procedures for the isolation of soluble type 1 collagen (COL1) require about 10 days from start to finish because of lengthy buffer incubations and laborious resuspensions of fibrils. Here, we describe a means to purify COL1 from small dermal biopsies in less than 3 hr.

피부에서 타입 I 콜라겐의 제조를위한 개선 된 방법

Soluble type 1 collagen (COL1) is used extensively as an adhesive substrate for cell cultures and as a cellular scaffold for regenerative applications. ...

Characterization of Complex Systems Using the Design of Experiments Approach: Transient Protein Expression in Tobacco as a Case Study

Plants provide multiple benefits for the production of biopharmaceuticals including low costs, scalability, and safety. Transient expression offers the additional advantage of short development and production times, but expression levels can vary significantly between batches thus giving rise to regulatory concerns in the context of good manufacturing practice. We used a design of experiments (DoE) approach to determine the impact of major factors such as regulatory elements in the expression construct, plant growth and development parameters, and the incubation conditions during expression, on the variability of expression between batches. We tested plants expressing a model anti-HIV monoclonal antibody (2G12) and a fluorescent marker protein (DsRed). We discuss the rationale for selecting certain properties of the model and identify its potential limitations. The general approach can easily be transferred to other problems because the principles of the model are broadly applicable: knowledge-based parameter selection, complexity reduction by splitting the initial problem into smaller modules, software-guided setup of optimal experiment combinations and step-wise design augmentation. Therefore, the methodology is not only useful for characterizing protein expression in plants but also for the investigation of other complex systems lacking a mechanistic description. The predictive equations describing the interconnectivity between parameters can be used to establish mechanistic models for other complex systems.

We describe a design of experiments approach that can be used to determine and model the influence of transgene regulatory elements, plant growth and development parameters, and incubation conditions on the transient expression of monoclonal antibodies and reporter proteins in plants.

사례 연구로 담배에 과도 단백질 발현 : 실험 접근의 디자인을 사용하여 복잡한 시스템의 특성

Plants provide multiple benefits for the production of biopharmaceuticals including low costs, scalability, and safety. Transient expression offers the ...

A Full Skin Defect Model to Evaluate Vascularization of Biomaterials In Vivo

Insufficient vascularization is considered to be one of the main factors limiting the clinical success of tissue-engineered constructs. In order to evaluate new strategies that aim at improving vascularization, reliable methods are required to make the in-growth of new blood vessels into bio-artificial scaffolds visible and quantify the results. Over the past couple of years, our group has introduced a full skin defect model that enables the direct visualization of blood vessels by transillumination and provides the possibility of quantification through digital segmentation. In this model, one surgically creates full skin defects in the back of mice and replaces them with the material tested. Molecules or cells of interest can also be incorporated in such materials to study their potential effect. After an observation time of one&#8217;s own choice, materials are explanted for evaluation. Bilateral wounds provide the possibility of making internal comparisons that minimize artifacts among individuals as well as of decreasing the number of animals needed for the study. In comparison to other approaches, our method offers a simple, reliable and cost effective analysis. We have implemented this model as a routine tool to perform high-resolution screening when testing vascularization of different biomaterials and bio-activation approaches.

A Full Skin Defect Model to Evaluate Vascularization of Biomaterials In Vivo

Vascularization is key to approaches in successful tissue engineering. Therefore, reliable technologies are required to evaluate the development of vascular networks in tissue-constructs. Here we present a simple and cost-effective method to visualize and quantify vascularization in vivo.

전체 피부 결함 모델은 생체의 혈관 신생을 평가하기<em&gt; 생체</em

Insufficient vascularization is considered to be one of the main factors limiting the clinical success of tissue-engineered constructs. In order to ...

Microscale Vortex-assisted Electroporator for Sequential Molecular Delivery

Electroporation has received increasing attention in the past years, because it is a very powerful technique for physically introducing non-permeant exogenous molecular probes into cells. This work reports a microfluidic electroporation platform capable of performing multiple molecule delivery to mammalian cells with precise and molecular-dependent parameter control. The system&#8217;s ability to isolate cells with uniform size distribution allows for less variation in electroporation efficiency per given electric field strength; hence enhanced sample viability. Moreover, its process visualization feature allows for observation of the fluorescent molecular uptake process in real-time, which permits prompt molecular delivery parameter adjustments in situ for efficiency enhancement. To show the vast capabilities of the reported platform, macromolecules with different sizes and electrical charges (e.g., Dextran with MW of 3,000 and 70,000 Da) were delivered to metastatic breast cancer cells with high delivery efficiencies (&#62;70%) for all tested molecules. The developed platform has proven its potential for use in the expansion of research fields where on-chip electroporation techniques can be beneficial.

A microfluidic vortex assisted electroporation platform was developed for sequential delivery of multiple molecules into identical cell populations with precise and independent dosage control. The system&#8217;s size based target cell purification step preceding electroporation aided to enhance molecular delivery efficiency and processed cell viability.

마이크로 소용돌이를 이용한 순차 분자 전달을위한 Electroporator을

Electroporation has received increasing attention in the past years, because it is a very powerful technique for physically introducing non-permeant ...

Development of a Multicellular Three-dimensional Organotypic Model of the Human Intestinal Mucosa Grown Under Microgravity

Because cells growing in a three-dimensional (3-D) environment have the potential to bridge many gaps of cell cultivation in 2-D environments (e.g., flasks or dishes). In fact, it is widely recognized that cells grown in flasks or dishes tend to de-differentiate and lose specialized features of the tissues from which they were derived. Currently, there are mainly two types of 3-D culture systems where the cells are seeded into scaffolds mimicking the native extracellular matrix (ECM): (a) static models and (b) models using bioreactors. The first breakthrough was the static 3-D models. 3-D models using bioreactors such as the rotating-wall-vessel (RWV) bioreactors are a more recent development. The original concept of the RWV bioreactors was developed at NASA&#39;s Johnson Space Center in the early 1990s and is believed to overcome the limitations of static models such as the development of hypoxic, necrotic cores. The RWV bioreactors might circumvent this problem by providing fluid dynamics that allow the efficient diffusion of nutrients and oxygen. These bioreactors consist of a rotator base that serves to support and rotate two different formats of culture vessels that differ by their aeration source type: (1) Slow Turning Lateral Vessels (STLVs) with a co-axial oxygenator in the center, or (2) High Aspect Ratio Vessels (HARVs) with oxygenation via a flat, silicone rubber gas transfer membrane. These vessels allow efficient gas transfer while avoiding bubble formation and consequent turbulence. These conditions result in laminar flow and minimal shear force that models reduced gravity (microgravity) inside the culture vessel. Here we describe the development of a multicellular 3-D organotypic model of the human intestinal mucosa composed of an intestinal epithelial cell line and primary human lymphocytes, endothelial cells and fibroblasts cultured under microgravity provided by the RWV bioreactor.

Cells growing in a three-dimensional (3-D) environment represent a marked improvement over cell cultivation in 2-D environments (e.g., flasks or dishes). Here we describe the development of a multicellular 3-D organotypic model of the human intestinal mucosa cultured under microgravity provided by rotating-wall-vessel (RWV) bioreactors.

인간의 장 점막의 다세포 3 차원의 Organotypic 모델의 개발은 미세 중력 하에서 성장

Because cells growing in a three-dimensional (3-D) environment have the potential to bridge many gaps of cell cultivation in 2-D environments (e.g., ...

Mesenchymal Stromal Cell Culture and Delivery in Autologous Conditions: A Smart Approach for Orthopedic Applications

Human Mesenchymal Stromal Cells (hMSCs) are cultured in vitro with different media. Limits on their use in clinical settings, however, mainly depend on potential biohazard and inflammation risks exerted by xenogeneic nutrients for their culture. Human derivatives or recombinant materials are the first choice candidates to reduce these reactions. Therefore, culture supplements and materials of autologous origin represent the best nutrients and the safest products.
Here, we describe a new protocol for the isolation and culture of bone marrow hMSCs in autologous conditions &#8212;&#160;namely,&#160;patient-derived serum as a supplement for the culture medium and fibrin as a scaffold for hMSC administration. Indeed, hMSC/fibrin clot constructs could be extremely useful for several clinical applications. In particular, we focus on their use in orthopedic surgery, where the fibrin clot derived from the donor&#39;s own blood allowed effective cell delivery and nutrient/waste exchanges. To ensure optimal safety conditions, it is of the utmost importance to avoid the risks of hMSC transformation and tissue overgrowth. For these reasons, the approach described in this paper also indicates a minimally ex vivo hMSC expansion, to reduce cell senescence and morphologic changes, and short-term osteo-differentiation before implantation, to induce osteogenic lineage specification, thus decreasing the risk of subsequent uncontrolled proliferation.

Culturing human Mesenchymal Stromal Cells (hMSCs) with autologous serum, reduces the risks of rejection by xenogeneic material and other negative effects. It also allows for the recovery of a subset of mesodermal progenitors, which can deliver fresh hMSCs. Embedding hMSCs in an autologous fibrin clot enables easy handling and effective surgical implantation.

중간 엽 기질자가 조건에서 세포 배양 배송 : 정형 외과 응용 프로그램에 대한 스마트 접근

Human Mesenchymal Stromal Cells (hMSCs) are cultured in vitro with different media. Limits on their use in clinical settings, however, mainly depend on ...

DNA Nanotubes as a Versatile Tool to Study Semiflexible Polymers

Mechanical properties of complex, polymer-based soft matter, such as cells or biopolymer networks, can be understood in neither the classical frame of flexible polymers nor of rigid rods. Underlying filaments remain outstretched due to their non-vanishing backbone stiffness, which is quantified via the persistence length (lp), but they are also subject to strong thermal fluctuations. Their finite bending stiffness leads to unique, non-trivial collective mechanics of bulk networks, enabling the formation of stable scaffolds at low volume fractions while providing large mesh sizes. This underlying principle is prevalent in nature (e.g., in cells or tissues), minimizing the high molecular content and thereby facilitating diffusive or active transport. Due to their biological implications and potential technological applications in biocompatible hydrogels, semiflexible polymers have been subject to considerable study. However, comprehensible investigations remained challenging since they relied on natural polymers, such as actin filaments, which are not freely tunable. Despite these limitations and due to the lack of synthetic, mechanically tunable, and semiflexible polymers, actin filaments were established as the common model system. A major limitation is that the central quantity lp cannot be freely tuned to study its impact on macroscopic bulk structures. This limitation was resolved by employing structurally programmable DNA nanotubes, enabling controlled alteration of the filament stiffness. They are formed through tile-based designs, where a discrete set of partially complementary strands hybridize in a ring structure with a discrete circumference. These rings feature sticky ends, enabling the effective polymerization into filaments several microns in length, and display similar polymerization kinetics as natural biopolymers. Due to their programmable mechanics, these tubes are versatile, novel tools to study the impact of lp on the single-molecule as well as the bulk scale. In contrast to actin filaments, they remain stable over weeks, without notable degeneration, and their handling is comparably straightforward.

Semiflexible polymers display unique mechanical properties that are extensively applied by living systems. However, systematic studies on biopolymers are limited since properties such as polymer rigidity are inaccessible. This manuscript describes how this limitation is circumvented by programmable DNA nanotubes, enabling experimental studies on the impact of filament rigidity.

Semiflexible 고분자 공부를 다양 한 도구로 DNA 나노튜브

Mechanical properties of complex, polymer-based soft matter, such as cells or biopolymer networks, can be understood in neither the classical frame of ...

Bioprinting of Cartilage and Skin Tissue Analogs Utilizing a Novel Passive Mixing Unit Technique for Bioink Precellularization

Bioprinting is a powerful technique for the rapid and reproducible fabrication of constructs for tissue engineering applications. In this study, both cartilage and skin analogs were fabricated after bioink pre-cellularization utilizing a novel passive mixing unit technique. This technique was developed with the aim to simplify the steps involved in the mixing of a cell suspension into a highly viscous bioink. The resolution of filaments deposited through bioprinting necessitates the assurance of uniformity in cell distribution prior to printing to avoid the deposition of regions without cells or retention of large cell clumps that can clog the needle. We demonstrate the ability to rapidly blend a cell suspension with a bioink prior to bioprinting of both cartilage and skin analogs. Both tissue analogs could be cultured for up to 4 weeks. Histological analysis demonstrated both cell viability and deposition of tissue specific extracellular matrix (ECM) markers such as glycosaminoglycans (GAGs) and collagen I respectively.

Cartilage and skin analogs were bioprinted using a nanocellulose-alginate based bioink. The bioinks were cellularized prior to printing via a single step passive mixing unit. The constructs were demonstrated to be uniformly cellularized, have high viability, and exhibit favorable markers of differentiation.

연골과 피부 조직 아날로그 소설 수동 Bioink Precellularization에 대 한 단위 기술 혼합을 이용 하 여 Bioprinting

Bioprinting is a powerful technique for the rapid and reproducible fabrication of constructs for tissue engineering applications. In this study, both ...

Recombinant Collagen I Peptide Microcarriers for Cell Expansion and Their Potential Use As Cell Delivery System in a Bioreactor Model

Tissue engineering is a promising field, focused on developing solutions for the increasing demand on tissues and organs regarding transplantation purposes. The process to generate such tissues is complex, and includes an appropriate combination of specific cell types, scaffolds, and physical or biochemical stimuli to guide cell growth and differentiation. Microcarriers represent an appealing tool to expand cells in a three-dimensional (3D) microenvironment, since they provide higher surface-to volume ratios and mimic more closely the in vivo situation compared to traditional two-dimensional methods. The vascular system, supplying oxygen and nutrients to the cells and ensuring waste removal, constitutes an important building block when generating engineered tissues. In fact, most constructs fail after being implanted due to lacking vascular support. In this study, we present a protocol for endothelial cell expansion on recombinant collagen-based microcarriers under dynamic conditions in spinner flask and bioreactors, and we explain how to determine in this setting cell viability and functionality. In addition, we propose a method for cell delivery for vascularization purposes without additional detachment steps necessary. Furthermore, we provide a strategy to evaluate the cell vascularization potential in a perfusion bioreactor on a decellularized&#160;biological matrix. We believe that the use of the presented methods could lead to the development of new cell-based therapies for a large range of tissue engineering applications in the clinical practice.

We propose a cell expansion protocol on macroporous microcarriers and their use as delivery system in a perfusion bioreactor to seed a decellularized tissue matrix. We also include different techniques to determine cell proliferation and viability of cells cultured on microcarriers. Furthermore, we demonstrate functionality of cells after bioreactor cultures.