RCCS生物反应器：重组蛋白制药的利器
Amgen and Genentech have established their positions as leaders in the biotechnology industry by producing recombinant protein drugs. The SCIDEMO.COM___NASA Rotary Cell Culture System technology is expected to increase the efficiency of producing such drugs at least ten fold.
回转式细胞培养系统_______生物制药进行大规模细胞培养
1.大量培养细胞的意义
动物和人的细胞可以合成有重要价值的蛋白质（例如干扰素、单克隆抗体等等），在科研、临床和商业上都有重大价值。要制备干扰素、单抗、尿激酶等，就需要大量培养哺乳动物的细胞、人的杂交瘤骨髓瘤细胞、人的肾细胞等等。虽然工业微生物技术已发展很完善，但是它主要是用于培养细菌类的细胞，它们有坚韧的细胞壁，对营养物质的要求较简单。而哺乳类细胞则大而脆弱，对营养物质要求高，不能游离生活，大多数要附着在某种表面才能生长。要大量培养哺乳类细胞是个困难的任务。
2.大量培养细胞的方法
小的细胞培养瓶，瓶底面积很小，只能培养很少细胞，要不断分瓶，得用许多长的瓶子不断滚动来获得较多的细胞。后来，人们又采取高密度培养方法，以求在一定的容积内能培养更多的细胞。对于要求附着生长的细胞，为提高表面积/体积的比值，用空心纤维和微载体作为附着物，可以大大提高培养容积的利用率。微珠作为载体，是提高培养密度的有效方法，细胞可以生长在微珠的表面，微载体法，可使在同样容积内培养细胞数增加几个量级。随着细胞的生长，附近的营养物质逐渐减少，废物逐渐增多，为了使生长的细胞不断接触到营养物质，并离开废物，微珠能在培养介质中分散和悬浮，并且不断供应培养液，不断排出废液。
3.适用范围
生物制药中进行大规模细胞培养
The only system for growing fragile human THREE-DIMENSIONAL CELLS AND TISSUE CULTURES
outside the human body. The research possibilities are endless, ranging from Cancer and HIV
to Tissue Engineering and the production of Pharmaceuticals.
发明于美国NASA的世界上唯一能在体外进行的“人的3维细胞/组织培养系统”RCCS™（Rotary Cell Culture System)已进入中国！RCCS™的应用面极广，从癌症和爱滋病研究，到组织工程和工业制药。 1.【RCCS™的机制和优势】 相比动态和静态组织培养系统，RCCS的主要优势是其能提供理想的环境，从而允许细胞聚集、3维生长和分化。这个优势使得我们的培养环境非常接近于体内。在大多数的动态培养系统中细胞/组织会受损，这主要是其与推进器接触而致。那么它为何会与推进器接触呢？因为悬浮，那悬浮是如何产生的？这归因于以下2个力：1.受到推进器产生的力；2.在培养的氧交换和悬浮过程中喷出的气泡所产生的 剪切力。在静态的平面培养瓶/培养皿中，2维的环境和塑料基质会改变基因表达和阻止分化。相比之下，在RCCS™中生长的细胞/组织因随机变化的重力矢量而悬浮，从而在培养液中成连续的自由落体状态。一个较好的比喻是：细胞从无限高的充满液体的管中落下。组织在培养液中会落下、翻滚和混合，受不到任何主宰生长方向的单个重力矢量的影响；组织会向各个方向生长。这就是RCCS™的机制。由于系统无推进器、空气升液器、气泡或搅拌器，使破坏性应力减到最小。因维持在一个理想的流体轨道中，故细胞能共同生长、互相交流和形成3维的结构。此外，RCCS™是目前唯一的能使研究者进行共同培养，而这种培养能提供本能的分化、高细胞增殖、低的细胞死亡率和增加细胞产物的分泌。总之，RCCS™的培养容器中充满了用作培养基的生长液以及细胞或组织材料。整个容器由电机驱动沿水平轴旋转。细胞颗粒在水平轴内建立均质的液体悬浮轨道。培养基以及细胞颗粒随容器一起旋转且不与容器壁和它物相撞。由于系统无推进器、空气升液器、气泡或搅拌器，使破坏性应力减到最小。RCCS中的细胞通过膜式气体交换器来吸氧和排出CO2。任何气泡都被清除，以防其旋涡对细胞的生长的影响。无破坏应力使生成的三维组织具有与父系相同的结构和功能。其组织的培养密度为10E10至10E11个细胞/ml；细胞的培养密度为10E8。至今所有的细胞组织都能培养。RCCS™具有普通培养装置无可比拟的三大特点:(1) 高分化度: RCCS使高分化的人体组织能在实验室中生长，模仿感兴趣的器官和肿瘤。 (2) 模拟微重力: 以前要在体外模拟正常组织的微环境因细胞外基质太复杂和难适应环境而受阻，而细胞外基质对于调节细胞骨架和细胞核基质蛋白非常重要。RCCS使分裂原本应在一起的细胞组织成分的重力问题得到解决。 (3) 三维细胞培养: 而普通生物反应器因要保持细胞的悬浮而产生剪切力。其破坏了细胞间和细胞与基质间的稳定，使组织和细胞集中于自身的修复, 而大大影响其生长和其它的正常生理功能。发孝罐主要培养大量细菌, 不适于大量培养人及其它哺乳动物细胞组织，因很难把大量的细胞移植到发孝罐中, 即使能够培养, 所得的细胞数也有限且很难生成组织。而RCCS行且可用于大规模生产，其细胞的成活率平均为97%且分化度极高。 2.【RCCS™的应用】 (在模拟的微重力环境下，细胞/组织的三维高分化的培养技术已进入临床和生产)组织移植：例如RCCS培养的肝组织和自然的在整体上无区别，使局部组织移植成为可能。疫苗生产：以前丙肝疫苗效果不佳的原因之一是用来产生这种疫苗的病毒并不长在人的肝脏中，现在RCCS培养的肝使产生肝炎疫苗的病毒生长在人的肝脏成为现实。RCCS在美国已广泛用于生产。 软骨再生：经培养的软骨密度极高，可治疗关节损伤。 ***、酶和其它由人体组织产生的蛋白以及基因工程：经培养的高分化的人体组织，其被刺激后能分泌治疗用的蛋白。例如经培养的神经组织产生的神经生长***能修复脊椎的损伤。 骨髓再生：培养的骨髓生长情况极佳，且可连续进行增生和低温冷冻保藏，可建大型骨髓库。 糖尿病：胰岛素培养后能插入体内继续生长，无数患者有望免去长期注射胰岛素。 有效杀灭肿瘤：对肿瘤组织活体取样，与自身的白细胞或淋巴细胞在RCCS中混合培养，刺激或驯化它们来识别和攻击肿瘤组织，然后把经驯化后有杀伤力的细胞直接注入病灶。这样，这些经感化的淋巴细胞彻底杀灭了肿瘤。 肿瘤、爱滋病、肾病和心脏病的理想模型：培养人的器官、腺体和淋巴结。然后感染这些类器官，再跟踪其生长。把药物用在被感染的经培养的组织模型上，研究其对抗疾病的效果及其对抗方式。例如肿瘤组织的培养有利于测试化疗药在病人自身的经培养和分化的肿瘤上的疗效，避免了用药的盲目性。以前这是在鼠的组织模型上进行的，但物种差异使许多肿瘤在鼠中的生长情况并不好。而在RCCS中，所有的肿瘤组织都完好生长，且避免了原先鼠蛋白的干扰。 皮肤移植：经培养的皮肤具有极高分化度避免了以前培养的皮肤在肌理、灵巧度和肤色的不足。 3.【我们所提供的各种培养系统的对比】 剪切力 物质传递 维数 异类细胞的共同培养 细胞密度 (细胞/ml)
静态培养 (T 型烧瓶) 无 准二维 3至4层的二维 有限 0.3 - 1x106
静态基质培养 无 有限的三维 有限的三维 有限 低
转瓶培养 中 好的二维 3至4层的二维 极有限 1 - 5x106
搅拌悬浮培养 中/高 好 极有限的三维 极有限 106 - 107
空气升液培养 中/低 好 极有限的三维 极有限 106 - 108
空心纤维灌注培养 无 好 有限的三维 有限 107 - 108
旋转培养 (生物反应器) 极低 极佳 出色的三维 极佳 108
RCCS™ Consists of a single station Rotator Base, Power Supply, Operation Manual, and Reusable STLV or HARV Culture Vessel of your choice.* Also accepts Disposable Vessels.
RCCS™-4 Consists of a four station Rotator Base, Power Supply, Operation Manual, and four Reusable STLV or HARV Culture Vessel of your choice - you may select a variety of vessels with the system. All vessels do not have to be the same.* Accepts Reusable and Disposable Vessels.
STLV Vessels Reusable Vessel. Oxygenated by a center core silicone rubber gas transfer membrane. The STLV is intended for microcarrier cell culture and explant tissue cultures.* Available in 55, 110, 250, or 500 ml sizes
HARV Vessels Reusable Vessel. Oxygenation by a flat silicone rubber gas transfer membrane. Used to culture both suspension and anchorage dependent cells.* Available in 50 and 10ml sizes
How It Works Rotary Cell Culture System
Since The Rotary Cell Culture System has no impellers, airlifts, bubbles,or agitators, tissue damage from impact and turbulence is significantly decreased as compared to conventional bioreactors. Shear stress and damage is so low that it is essentially insignificant.
The operation of Synthecon''s RCCS differs from all other cell culture systems.The cylindrical culture vessel is filled with culture fluid and the cells or tissue particlesare added. All air bubbles are removed from the culture vessel. The vessel is attached to the rotator base and rotated about its horizontal axis. Tissues, cells or cell aggregates establish a fluid orbit within the culture medium in the horizontally rotating cylindrical vessel. The aggregates remain suspended in the culture medium having
little or no contact with the vessel wall.
A schematic view of the Perfused System, displaying medium vessel infusion mode. Perfuses media and nutrients in a single pass through the culture chamber from a media reservoir to waste. Also mode for refreshing media in the Recirculation mode.
As organoids grow in size, the rotational speed is adjusted to compensate for the increased settling rates of the larger particles. The motion of the vessel is sufficient to exchange nutrients, wastes and dissolved gases, and to allow cell-to-cell contacts that lead to aggregation.
Oxygen supply and carbon dioxide removal is achieved through a gas permeable silicone rubber membrane, which acts very much like the membranes of the lung. Since The Rotary Cell Culture System has no impellers, airlifts, bubbles, or agitators, tissue damage from impact and turbulence is significantly decreased as compared to conventional bioreactors. Shear stress and damage is so low that it is essentially insignificant. Under these conditions, cells communicate and synthesize extra cellular matrix as they would in vivo.
A schematic view of the Perfused System, displaying syringe-injection mode. In this mode flow is open to waste to compensate for displacement during injection. Allows for addition of reagents to supplement media.
Unlike cell and tissue cultures grown in two-dimensional flat plate systems, organoids grown in the Rotary Cell Culture System are functionally similar to tissues in the human body. You will be able to grow three-dimensional tissues in vitro that mimic the structure and function of the same tissue in vivo.
Tissue explants from biopsies or surgical procedures can be placed directly into the RCCS
maintaining their original cyto-architecture or can be expanded into tissue "organoids" or tumor models. This has important value in such fields as bone marrow, skin, pancreas and liver regeneration and the potential testing of chemotherapy drugs on the patient''s own tumor material ex vivo.
A schematic view of the Perfused System, displaying closed recirculation mode. Continuous flow of media and nutrients through a closed loop system, from the culture chamber advanced by the peristaltic pump, oxygenated via external silicone oxygenator and back into the culture chamber.
STLV - Exploded View Oxygenated by a center core membraneoxygenator, these reusable vessels areused for microcarrier cell cultureand explant tissue cultures.Shown here: exploded view STLV - Assembled Oxygenated by a center core membraneoxygenator, these reusable vessels areused for microcarrier cell cultureand explant tissue cultures.Shown here: assembled
Advantages and Benefits of the Rotary Cell Culture System
The primary advantage of the RCCS over either dynamic or static tissue culture systems is that its gentle environment allows cells to aggregate, grow 3-dimensionally, and differentiate.
This advantage results in cells or tissues that very closely resemble the in vivo tissue quivalent.
In most dynamic systems, cells or tissues are suspend by physical forces that result in injury to the cells from contact with an impeller or by the shear forces caused by bubbles being sparged up through the culture for oxygenation and suspension.
These conditions cause injury to cells or tissues.
In static flat culture flasks or dishes the 2-dimensional environment and plastic substrate tend to alter gene expression and prevent differentiation.
Conversely, cells or tissues grown in the Rotary Cell Culture System br> are suspended by randomizing the gravity vector so they are, in effect, in continuous free-fall through the culture medium.
A good analogy would be to imagine a cell falling through a fluid-filled tube of infinite height. The tissue would fall, tumble, mix in the media and without any single gravity vector dictating growth direction; the tissue would grow in all directions. That is mechanism of the RCCS
Destructive stress forces are insignificant because this system has no propellers, airlifts, bubbles, or agitators. Because the cells are maintained in a gentle fluid orbit, the cells are allowed to co-localize, communicate with each other and form three-dimensional structures.
Furthermore, the RCCS is the only system currently available that allows researchers to introduce co-cultures that provide spontaneous tissue differentiation, higher cell multiplication, decreased cell death, and increased secretion of cell products.
Product Benefits
More than 50 cell and tissue types have been successfully cultured in the RCCS
Cells not only grow, but also develop into functional specialized tissue organoids. Scientists from academia, industry and government have published over 70 peer-reviewed articles utilizing RCCS technology the in scientific literature. These publications clearly show that the RCCS is the best technology available for growing differentiated mammalian tissue in vitro.
Benefits Include:
Wide Range of RCCS
Equipment Available, Within Any Laboratory Budget Three-Dimensional Tissues that Mimic Human Tissue in the Body Growth of Valuable Bio-Products from Tissue Reduced Costs of Culture Materials Simple Operating Procedures
Low Capital Investment Easy To Learn and Use Consistency of Results High-Density Cultures Rapid Culture Growth Harvesting Efficiency Wide Range of Uses Labor savings
Featured Tissues Grown Using the Rotary Cell Culture System
Brain
Neurons are being cultured in the RCCS to enable researchers to study various neurological diseases, e.g., Parkinson''s and Alzheimer''s.
Cancer Research
Research and phase 1 clinical trials are being conducted using the RCCS to investigate the growth and malignant changes of cancer as they develop from single cells to tumors including melanoma, prostate, breast and ovarian cancers, osteosarcoma, glioma, and colon cancers.
The RCCS model of three-dimensional cell culture enables examination of gene expression as a function of the stage of tumor cell aggregate growth. The Rotary Cell Culture System
is also being used in studies involving clinical cancer drug sensitivity testing,chemotherapy drug development, and cancer vaccine development.
Cartilage and Bone
Several institutions are investigating methods for regenerating and repairing bone and cartilage, including bone implants and grafts. These methods show promise for providing a therapy for skeletal disorders, and for allowing doctors to repair bone injuries or perform reconstructive surgery.
The RCCS creates a culture environment conducive to cell aggregation, and provides a powerful new tool for the study of bone physiology, biochemistry and gene expression.
Heart
Research also is being conducted to grow heart cells. In a specialized application televised recently on PBS, heart cells were shown growing in the RCCS bioreactor and actually beating under a microscope. This ability to grow heart tissue can greatly benefit open-heart surgeons who have to cut the pericardium. Often during healing, thepericardial sac actually adheres to the sternum.
Doctors who are using the RCCS are working towards creating a heart tissue "patch" to cover that wound thus eliminating adhesion to other organs.
Kidney
Kidney cells cultured in the RCCS bioreactor are being used for toxicity models and drug testing.
Liver
The RCCS provides a means to grow and expand hepatocyte cultures into high fidelity models of liver tissue. Liver tissue models will have a number of important applications including the design of gene therapy protocols, hepatocyte transplantation, the development of extra corporal assist devices, and test systems for the design of hepatitis vaccines and test of antiviral compounds.
In addition, researchers are conducting metabolism studies to determine how liver cells react to and metabolize toxins. Long-term hepatocyte cultures are also being maintained.
In conventional cultures, hepatocytes cannot be maintained for more than 10 to 15 days. Investigators using the RCCS however, have routinely maintained hepatocytes in vitro for as long as 70 days.
Pharmaceuticals
Valuable biopharmaceutical proteins for use in drugs are being produced in significantly higher quantities per cell using the RCCS
As a result, the need for larger, more costly bioreactors is reduced, as well as the volume of expensive materials that are required for protein production, and labor costs are significantly lowered.
Stomach
The RCCS bioreactor has helped create a gut model that''s being used in gastrointestinal absorption studies with biopharmaceuticals.
Tissue Explants
The U.S. Army Medical Research Institute of Infectious Diseases at Fort Detrick, Maryland, is using RCCS explants of human liver, spleen, and lymph nodes in an effort to find new compounds that could be used to treat patients infected with the lethal Ebola virus.
In other experiments, the RCCS has been able to preserve the normal three-dimensional structure of explanted human prostate tissue in culture. After an initial period of reorganization, explanted blocks of prostate tissue have been shown to maintain both tubuloglandular and fibromuscular stromal elements during six weeks of continuous RCCS culture. When added to the culture medium
of the bioreactor, malignant prostate epithelial cells can attach to and invade the normal fibromuscular stroma of the tissue. This provides a useful model for the study of host-tumor cell interactions in the human prostate.
The Future
SYNTHECON is growing smartly and strategically.
As the world''s leading manufacturer of 3-dimensional tissue culture systems, SYNTHECON is moving swiftly to expand awareness and demand throughout the scientific community of its leading-edge technology. And, it is committed to continually refining and improving its rotating cell culture systems for even broader applications.
Applications of the Rotary Cell Culture System
Scientists and clinicians worldwide are using the RCCS bioreactors for a wide variety of applications.
They are growing normal human tissue in vitro for testing of therapeutic drugs or for growing replacement tissue such as liver, skin, bone marrow, cartilage, heart muscle, and other tissues.
The RCCS bioreactor provides the research community with an excellent in vitro environment for culturing cells such as human tumor, viruses and cells that produce valuable bio-products including proteins, enzymes, hormones, antibodies and antigens.
Culture conditions in the RCCS also provide a superior in vitro environment for studying the effects of micro-environmental cues, especially intercellular communication on tissue-specific cell assembly, differentiation and function, and for studying phenotypic instability, drug/radiation-resistance, and the inter and intracellular signaling pathways involved in clonal expansion.
Synthecon''s RCCS bioreactors are being used at a number of prestigious research facilities.
The Cell & Gene Therapy Institute in San Antonio is using RCCS bioreactors to provide an inexhaustible supply of HIV infected human tissue for the development of HIV vaccines and test HIV pharmaceuticals.
The Food and Drug Administration uses the RCCS for intestinal parasite research.
The U.S. Army Medical Research Institute of Infectious Diseases at Ft. Detrick, Md.
has used the RCCS to grow Hong Kong Avian flu, Ebola, Monkey pox, Marbung and Hanta viruses in an effort to develop cures.