Bài giảng kỹ thuật cấy chuyền và tạo dòng [chuẩn nhất]

Đại học Quốc gia TP HCM Trường Đại học Khoa học Tự Nhiên Viện Tế bào gốc

KỸ THUẬT CẤY CHUYỀN VÀ TẠO DÒNG

CẤY CHUYỀN?

Passage / Subculture

cell density the

CẤY CHUYỀN

Khi nào thì cần cấy chuyền?

When (cells/cm2 substrate) reaches a level such that all of the available substrate is occupied, or when the cell concentration (cells/mL medium) exceeds the capacity of the medium, growth ceases or is greatly reduced.

CẤY CHUYỀN

Khi nào thì cần cấy chuyền?

Cell Confluency (source: https://www.mirusbio.com/transfectopedia/methods)

NUÔI CẤY SƠ CẤP

NUÔI CẤY THỨ CẤP

Nuôi cấy thứ cấp là nuôi cấy tế bào được tính từ sau lần cấy chuyền đầu tiên

Gentile, P., et al. (2012) Siennicka, K., et al. (2016)

fraction) SVF (stromal vascular trước khi được đưa vào nuôi cấy là một phức hợp gồm tế bào máu ngoại vi, MSC, tế bào tiền thân nội mô, tế bào cơ trơn, tế bào quanh mạch (pericyte), nguyên bào sợi, dưỡng bào, tế bào tiền thân tạo mỡ,… [38, 115]. Sau vài thế hệ cấy chuyền SVF trong điều kiện tiêu chuẩn, quần thể tế bào trở nên khá tương đồng của tế bào trung mô – ASC [38].

CẤY CHUYỀN Các bước cơ bản của cấy chuyền

• HÚT BỎ MÔI TRƯỜNG NUÔI CẤY CŨ

• RỬA BỀ MẶT GIÁ THỂ NUÔI (CÓ TẾ BÀO BÁM DÍNH)

• TÁCH CÁC TẾ BÀO BÁM KHỎI BỀ MẶT DỤNG CỤ NUÔI

• PHA LOÃNG CÁC TẾ BÀO BẰNG MÔI TRƯỜNG MỚI

Subculture of Monolayer. Stages in the subculture and growth cycle of monolayer cells after trypsinization

Cấy chuyền tế bào bám dính

Loại bỏ môi trường khỏi bình nuôi Rửa bề mặt bình nuôi với PBS- (free Ca2+ Mg2+)

Bổ sung dung dịch trypsin/EDTA (0,5- 3ml/bình) Ủ trong tủ ấm 370C

Khi tế bào co tròn, nổi lên tiến hành bổ sung một lượng môi trường có chứa huyết thanh Rửa loại bỏ trypsin bằng cách ly tâm 1000- 3000 vòng/phút

Tái huyền phù và pha loãng theo tỉ lệ thích hợp

Expansion of Mesenchymal Stem Cells

1.Subculture cells once they have reached approximately 80% confluence and are actively proliferating. IMPORTANT: Subculture cells before reaching 100% confluency. 2.Carefully remove the medium from the 10-cm tissue culture plate containing the confluent layer of human mesenchymal stem cells. Apply 3-5 mL of Trypsin-EDTA Solution (SM-2003-C) and incubate in a 37°C incubator for 3-5 minutes. 3.Inspect the plate and ensure the complete detachment of cells by gently tapping the side of the plate with the palm of your hand. 4.Add 5 mL Mesenchymal Stem Cell Expansion Medium (SCM015 or SCM045) to the plate. 5.Gently rotate the plate to mix the cell suspension. Transfer the dissociated cells to a 15 mL conical tube. 6.Centrifuge the tube at 300 x g for 3-5 minutes to pellet the cells. 7.Discard the supernatant 8.Apply 2 mL Mesenchymal Stem Cell Expansion Medium (pre-warmed to 37°C) containing 8 ng/mL FGF-2 to the conical tube and resuspend the cells thoroughly. IMPORTANT: Do not vortex the cells. 9. Count the number of cells using a hemocytometer. 10.Plate the cells at a density of 5,000-6,000 cells per cm2 into the appropriate flasks, plates, or wells in Mesenchymal Stem Cell Expansion Medium containing 8 ng/mL FGF-2. 11.Cells can be frozen in MSC growth media plus 10% DMSO (D2650) at a density of 2X106 cells/vial.

CẤY CHUYỀN

1. Trypsin/EDTA 2. EDTA (EthyleneDiamineTetraAcetic) 3. Scrape 4. …

Các phương pháp tách tế bào lên khỏi bề mặt dụng cụ nuôi

CẤY CHUYỀN Vai trò của trypsin/EDTA

CẤY CHUYỀN Vai trò của EDTA

 aminopolycarboxylic acid, colourless, water-soluble solid  able to "sequester" metal ions such as Ca2+ and Mg2+

C10H16N2O8

CẤY CHUYỀN Scrape

Scraper

CẤY CHUYỀN Các tiêu chí trong quá trình cấy chuyền

1. Density of Culture

CẤY CHUYỀN Các tiêu chí trong quá trình cấy chuyền

2. Exhaustion of Medium

CẤY CHUYỀN Các tiêu chí trong quá trình cấy chuyền

3. Time Since Last Subculture

Ideally, a cell concentration should be found that allows for the cells to be subcultured after 7 days, with the medium being changed after 3–4 days. (Culture of Animal Cells: A Manual of Basic Technique and Specialized Applications, Sixth Edition)

CẤY CHUYỀN Các tiêu chí trong quá trình cấy chuyền

4. Requirements for Other Procedures

(1) cells should not be subcultured while still

within the lag period

(2) cells should always be taken between the middle of the log phase and the time before which they have entered the plateau phase of a previous subculture

CẤY CHUYỀN Cell Concentration at Subculture

As a general rule, most continuous cell lines subculture satisfactorily at a seeding concentration of between 1 × 10^4 and 5 × 10^4 cells/mL

Spinner bioreactor

Fig. 13.5. Stirrer Culture. A small stirrer flask, based on the Techne design, with a capacity of 250–1000 mL. The cell suspension is stirred by a pendulum, which rotates in an annular depression in the base of the flask.

CẤY CHUYỀN Propagation in Suspension

 trypsin treatment is not required so subculture is quicker and less traumatic for the cells

 replacement of the medium (feeding) is not usually carried out with suspension cultures

 scale-up is easier

passage number / generation number

The passage number is the number of times that the culture has been subcultured, whereas the generation number is the number of doublings that the cell population has undergone, given that the number of doublings in the primary culture is very approximate.

Serial subculture and the relationship number, passage between generation number and split ratio. (Figure modified from Fig. 12.5, Culture of Animal Cells, 4th ed, Freshney (2000) and reproduced by permission of Wiley-Liss, a division of John Wiley & Sons.)

TẠO DÒNG? Cloning

Cell line / Cell Clone / Cell lineage / Cell Strain

Once a primary culture is subcultured (or passaged), it becomes known as a cell line. This term implies the presence of several cell lineages of either similar or distinct phenotypes. If one cell lineage is selected, by cloning (see Protocol 14.1), by physical cell separation (see Chapter 15), or by any other selection technique, to have certain specific properties that have been identified in the bulk of the cells in the culture, this cell line becomes known as a cell strain.

Cell lineage denotes the developmental history of a tissue or organ from the fertilized embryo. Cell lineage can be studied by marking a cell (with fluorescent molecules or other traceable markers) and following its progeny after cell division.

TẠO DÒNG? Cloning

Cloning is to isolate pure cell strains or cloning may help to reduce the heterogeneity of a culture

continuous cell line / continuous cell strain

If a cell line transforms in vitro, it gives rise to a continuous cell line, and if selected or cloned and characterized, it is known as a continuous cell strain.

Dilution Cloning

Cloning in Microtitration Plates

Cloning in Suspension

Some cells, particularly hematopoietic stem cells and virally transformed fibroblasts, clone readily in suspension. To hold the colony together and prevent mixing, the cells are suspended in agar or Methocel and plated on an agar underlay or into dishes that need not be treated for tissue culture.

(or may

trypsinized

to e

Feeder Layers. Cells are irradiated and be trypsinized first and then irradiated in suspension, or treated with mitomycin C) and seeded at a low density cloning enhanc efficiency. (Photo courtesy of M. G. Freshney.)