BMB206: 3D spheroid culture as long term in vitro cell culture model (5 ECTS)

STADS: 01010101

Level
PhD course

Teaching period
The course is offered in the spring semester.

Teacher responsible
Email: kwr@bmb.sdu.dk

Additional teachers
sjf@bmb.sdu.dk

Timetable
Show entire timetable
Show personal time table for this course.

Comment:
Kurset bliver afholdt én uge i forårssemesteret (april, maj). Den aktuelle dato er fleksibel og kan tilpasses efter andre aktiviteter på BMB (fx. antallet af kurser som udbydes til de studerende i det givende tidsrum)

Prerequisites:
None

Academic preconditions:
This is a postgraduate level course (master students, PhD level)
The participants will be expected to have knowledge about basic cell physiology and biochemistry on a bachelor level. Knowledge of the basic principles of light microscopy techniques will be useful but not required. Due to available laboratory space, the number of course participants is limited to maximum six persons

Course introduction
Since the breakthroughs that were made in cell culture in the 1950’s (in relation to the development of the polio vaccine), the prime focus has been on how to successfully grow and propagate cells in culture. Traditional monolayer cell culture techniques (2 dimensional cell culture = 2D) focused on cell growth conditions promoting a rapidly growing, but low differentiated cells. Cell cultured under such conditions are usually “locked” in a state where they are not able to expound their advanced cellular functions. In other words, cells lose many of their physiological attributes important for organ function when grown in classical 2D cell culture techniques.
It is becoming widely realised that new cell culture techniques focused on cell functionality are needed. Evidence is emerging that if cells are grown under the correct conditions, they automatically organise themselves into aggregates which have features resembling the adult mature tissue from which they came. They also gain in vivo physiological functionality.
Three dimensional (3D) structures appear to be essential for in vivo functionality [1-3]. Endothelial cell spheroids can from a hollow monolayer which expresses a tissue-like phenotype [4], human epidermal cells undergo proliferative differentiation and form a contact-lens-like disk with all the in vivo EM features of skin (except hair) [5], canine epithelial kidney cells form cysts which resemble rudimentary kidney glomerulae [6] or when treated with a growth factor differentiate into branched tubules as seen in the kidney [7]. Despite this, most research is carried out on cells, grown in classical flat (2D) culture systems.
During this course, the students will focus on one of the new 3D culture techniques, which utilise the growth of 3D cell spheroids in a micro-gravity environment. The students will learn how to manage the 3D cultures, use different culture quality validation methods, as well as how to characterise and prepare biological samples for further studies (sample preparation for further applications)
The course will also give the knowledge about traditional 2D techniques, specific requirements for cell culture laboratories (laboratory safety classification), describe and demonstrate in use the electronic laboratory management as part of Good Laboratory Practice (GLP)

References
[1] L.G. Griffith, M.A. Swartz, Capturing complex 3D tissue physiology in vitro. Nat Rev Mol Cell Biol 7 (2006) 211-224.
[2] F. Pampaloni, E.G. Reynaud, E.H. Stelzer, The third dimension bridges the gap between cell culture and live tissue. Nat Rev Mol Cell Biol 8 (2007) 839-845.
[3] L.J. Nelson, S.W. Walker, P.C. Hayes, J.N. Plevris, Low-shear modelled microgravity environment maintains morphology and differentiated functionality of primary porcine hepatocyte cultures. Cells Tissues Organs 192 (2010) 125-140
[4] M.J. Bissell, A. Rizki, I.S. Mian, Tissue architecture: the ultimate regulator of breast epithelial function. Curr Opin Cell Biol 15 (2003) 753-762.
[5] P.K. Jensen, S.J. Fey, P.M. Larsen, J.O. Norgard, L. Bolund, Morphological differentiation and changes in polypeptide synthesis pattern during regeneration of human epidermal tissue developed in vitro. Differentiation 47 (1991) 37-48.
[6] L.E. O'Brien, M.M. Zegers, K.E. Mostov, Opinion: Building epithelial architecture: insights from three-dimensional culture models. Nat Rev Mol Cell Biol 3 (2002) 531-537.
[7] A.L. Pollack, G. Apodaca, K.E. Mostov, Hepatocyte growth factor induces MDCK cell morphogenesis without causing loss of tight junction functional integrity. Am J Physiol Cell Physiol 286 (2004) C482-494.

Qualifications
After the course, the course participant’s should be able to:
- use the 3D spheroid cell culture system
- use material and interpreted result produced with us of the 3D spheroid cell culture system
 - calculate the doubling time of cells grown in 3D cell cultures
 - utilise luminescence-based assays for cell welfare assessment
 - use light microscopy techniques for culture quality assessment
 - use the principles of an electronic laboratory logbook for experiment documentation and data transfer

Expected learning outcome
After the course, the course participant’s will know:
- basic knowledge about general cell culture techniques
- in depth knowledge about the 3D spheroid cell culture system
- laboratory classification
- types of laboratory documentation

Subject overview
1. General techniques and working habits in cell culture laboratory
1.1. What is a cell and tissue culture laboratory:
- laboratory classification
- what is needed in a cell and tissue culture lab
- good cell laboratory practice
- laboratory documentation
- thinking like a cell
1.2 Commonly used techniques for cell culture growth
- classical cell culture (flat, 2D) on plates, flasks, multi-well plates
- cell validation: vitality, morphology, counting, population assessment, passage number
- trypsinisation (when and when not to)
- commonly used media and why they are used (glucose, galactose, serum free)
- suspension cultures (rotation flasks, mixing containers)
- static 3D cultures (scaffolds, gel sandwich cultures)

2: 3D spheroid culture in use.
2.1. Research based examples of 3D cell culture usage.
- Literature based examples of use of rotary cell culture vessel system (primarily Synthecon vessels)
-TCEL group experiment based examples (published results and on-going experiments)
2.2. Preparation for bioreactor handling.
Introduction to specific operational techniques used with the bioreactor vessels. Presentation of techniques used for sample handling and analysis.
2.3 In detail presentation of our bioreactor system
 - theoretical principle explanation
 - hardware presentation
 - operational modes
2.4 Theoretical aspects of 3D cell spheroid growth in bioreactor vessels
 - importance of correct rotation speed control
 - influence of population size on individual spheroid growth
 - general maintenance procedures
 - sampling procedures (what we can sample and what we should be aware of)

Literature
There isn't any litterature for the course at the moment.

Website
This course uses e-learn (blackboard).

Prerequisites for participating in the exam
None

Assessment and marking:
Oral exam, Danish 7mark scale, internal examiner. The oral exam takes place one week after the end of Laboratory excercises. (01010102)Laboratory exercise, pass/fail, internal evaluation by teacher. (01010122)
Project report, pass/fail, internal evaluation by teacher. (01010112)

The student must have passed laboratory exercises and the project report ind order to be able to take part in the oral exam.

The students will be evaluated based on their project description report and oral exam.
For the examination the Electronic Laboratory Logbook (ELL) and the Electronic Experiment Protocol (EEP) will be evaluated for its usefulness and completeness as prepublication materials.
Before the oral examination, students will receive written feedback for their ELL and EEP’s. The oral examination will consist of a general cell culture knowledge discussion (covering the subjects in the course) and specific project discussion based on the ELL and EPP provided by the Student and Teacher Feedback document provided by the teacher.

Expected working hours
The teaching method is based on three phase model.
Intro phase: 40 hours
Skills training phase: 29 hours, hereof:
 - Tutorials: 5 hours
 - Laboratory exercises: 24 hours

Educational activities

Language
This course is taught in English.

Remarks
This is a 1-week block course, which will be run during the spring semester (April, May) the exact date is flexible and can be adjusted to the general amount of activities at the BMB (e.g. number of courses offered to the students in particular time)

Course enrollment
See deadline of enrolment.

Tuition fees for single courses
See fees for single courses.