Organotial Human laryngeal carcinoma organoid Culture Kit,Absin_specification/price/image

Product Detail
Company Profile

Product Specification

Synonym

Carcinoma of the larynx Organoid Culture Kit

Description

Component	Size
Human laryngeal cancer organoid culture Medium A	100mL
Original generation of cultivating buffer B	250mL
Primitive organization digestive juices C throat cancer	30mL
Organoid passage digest D	30mL
Tissue preservation solution E	100mL
Organoid cryopreservation solution F	20mL
Organ class subculture buffer G	250mL

Isotype

IgG

General Notes

1. Human laryngeal cancer organoid culture medium A can be stored at 4 ℃ for 3 months. It is recommended to use it within 1 month after receiving goods at 4 ℃, and it is not recommended to store it at -20 ℃ for a long time to avoid repeated freezing and thawing more than 2 times.
2. Tissue preservation solution E and human laryngeal cancer primary tissue digestion solution C contain nutrients to maintain cell activity. In order to maintain the activity of nutrients, it is not recommended to store at -20 ℃ for a long time to avoid repeated freezing and thawing more than 2 times.
3. In 2– The matrix glue was thawed overnight at 8 ° C ambient conditions. When using matrix glue, place it in an ice box to prevent premature solidification. The matrix glue forms a gel within 20 min at 37 ° C.

Instructions

Taking 24-well plate as an example
I. Sample pretreatment
1. Experimental materials: primary culture buffer B (precooled at 4 ℃), tissue preservation solution E, sampling tube, tissue transport box, ice pack
2. Sample sampling and transportation: the tissue should be placed in tissue preservation solution E within 30 min in vitro, the primary culture buffer B should be washed 3-5 times, the blood on the surface of the tissue should be washed, put into tissue preservation solution E, and the sampling tube should be stored and transported at 4 ℃ for 72 hours.

II. Primary Culture of organoids
1. Experimental materials:
(1) Materials to be prepared: Matrix glue (melt in 4 ℃ refrigerator 24 h in advance), tweezers (10 cm), pointed ophthalmic surgical scissors/surgical blade, disposable 60 mm Petri dish, 1.5 mL/15 mL/50 mL EP tube, 100 um cell filter, 3 mL Papanicolaou straw /1000 uL pipettes gun, 24-well cell culture plate, metal ice box.
(2) Kit reagents: Primary culture buffer B (4 ° C), primary tissue digestion buffer C (37 ° C), human laryngeal cancer organoid medium A (room temperature or 37 ° C).
2. Organoid construction:
(1) Laryngeal cancer tissues after sampling are recommended to be stored and transported at 2-8 ℃, and quickly transported to a clean laboratory for the experimental process of human laryngeal cancer organoid construction. The tissues are taken photos and the detailed information is registered.
(2) After the sampling tube was disinfected, the tissue was removed on the ultra-clean table and placed in the culture dish. The primary culture buffer B was added, and the cleaning operation was repeated for 3 or more times with a 3 mL papanicolaher pipette or 1000 uL pipetting knife.
(3) Remove the tissue impurities with ophthalmic scissors or surgical blade, transfer the forceps to 1.5 mL EP tube, further mechanically dissociate the tissue into a volume of 1-3 mm³tissue block with ophthalmic scissors, and transfer to 15 mL EP tube. Then 5 mL of human laryngeal cancer tissue digestion solution was added and digested at 37 ℃ for 15-25 min. During the digestion process, the tissue digestion was observed under the microscope every 10 min. A small amount of digestive fluid was taken for observation under the microscope, and more cell clusters or single cells below 70 um were observed before the next operation. Note: If the amount of tissue was too small or the biopsy tissue was digested with 1 mL of human laryngeal cancer primary tissue digestion solution C in 1.5 mL EP tube.
(4) After digestion, the tissues were filtered through a 100 um pore size cell screen, and the filtrate was collected. The digestion was terminated by rinsing with 3 times the volume of primary culture buffer B. The supernatant was discarded after enrichment and centrifugation at 300 g for 5 min. If the cell precipitate contained red blood cells, 1-2 mL red blood cell lysate was added for 1-2 min, diluted to 10 mL, and the supernatant was discarded after enrichment and centrifugation at 300 g for 5 min. Note: when there is too little precipitation or no red blood cells, the tumor organoid culture is performed directly.
(5) The volume of cell precipitates collected by centrifugation was observed and resuspended by adding 25 times the volume of matrix glue to form a 3D culture space structure, and bubbles were avoided during the resuspension process. The volume of cell precipitation is shown in the figure below, and 200 uL, 150 uL, 100 uL and 50 uL of matrix glue were added respectively according to the amount of cell precipitation in the figure.

example: 24 hole cell culture plate according to the uL 25-30 uL/hole point glue, matrix all at 0 to 4 & have spent operation under the condition of ℃. The cell culture plate was placed in A 37 ° C incubator for 10-15 min, and after the matrix gel had solidified, 500-750 uL of human laryngeal cancer organoid medium A (preheated at 37 ° C) was added to each well for culture.

III. Organoid subculture
1. Experimental materials:
(1) Materials to be prepared: matrix glue (melt in 4 ℃ refrigerator 24 hours in advance), 1.5 mL/15 mL EP tube, 3 mL Papanicolaek pipette /1000 uL pipette gun, 24-well cell culture plate, metal ice box.
(2) Kit reagent: organoid passage culture buffer G (4 ℃), organoid passage digestion solution D (37 ℃), human laryngeal cancer organoid culture medium A (room temperature or 37 ℃).
2. Organoid passage:
(1) Select appropriate organoids for passage, generally grow for about a week, more than 20 organoids can be seen under a microscope at 10X, or organoids with a size of 100-200 um. The same volume of organoid passage culture buffer G was added to each well. The matrix glue was gently blown off by a pipetting gun, collected in 15 mL EP tubes, transferred to an EP tube every 6-8 Wells, and left for 10-15 min at 4 ° C.
(2) Determine whether digestion and passage are necessary according to the growth of the organoids. Note: After centrifugation, if there is less precipitation at the bottom of the tube, no cells are seen, and the matrix glue is not stratified, it can be resuspended again, the centrifugal force can be increased, and then centrifuged again.
a: When the number of organoids was insufficient or the volume was small, the supernatant was discarded by centrifugation at 300 g for 5 min.
b: When the number of organoids is large or large, 300 g centrifuge 5 The supernatant was discarded in min and either digested with digester or mechanically.
Digestion with digestive fluid: Add 1-2 mL organoid passage digestion solution D, blow off the cell precipitate, digest at room temperature for 2-4 min, blow every minute, observe under the microscope, until the digestion to the state (FIG. A-B) can be stopped. The digestion was terminated by adding 3 times the volume of organoid digestion liquid in organoid passage culture buffer G, and the supernatant was discarded by centrifugation at 300 g for 5 min.
Mechanical digestion: Add 1 mL organoid passage culture buffer G, blow with 1 mL pipettes for 20-30 times until blowing to (FIG. C-D) state, add 10 mL passage buffer, centrifugation at 300 g for 5 min and discard the supernatant.

(3) Observe the volume of organoid precipitation collected by centrifugation. If the precipitation is very small, 1 times the volume of precipitation supernatant can be reserved for gelatin injection, a lot of precipitation supernatant can be absorbed, add 25 times the volume of organoid precipitation matrix glue to resuspend the organoid. Example: The 24-well cell culture plate was dispensed at 25 ul-30 uL/ well, and the matrix glue was maintained at 0-4 ℃ throughout the operation . The cell culture plate was placed in A 37 ℃ incubator for 10-15 min, and after the matrix gel solidified, 500-750 uL of human laryngeal cancer organoid medium A (preheated at 37 ℃) was added to each well for culture.

IV. Organoid Cryopreservation
1. Experimental materials:
(1) Self-supplied materials: 15 mL EP tube, cell cryopreservation tube, program cooling box, pipeting gun.
(2) Kit reagent: organoid passage culture buffer G (4 ℃), organoid freezing solution F.
Organoid cryopreservation:
organoids not in use should be frozen and stored in a low temperature environment.
(1) The medium was sucked out, and the equal volume of organoid passage culture buffer G was added to each well. The matrix glue was gently blown off by a pipetting gun, collected in 15 mLEP tubes, transferred to an EP tube every 6-8 Wells, and left for 10-15 min at 4 ℃. The supernatant was discarded by centrifugation at 300 g for 5 min. Then 2 mL organoid cryopreservation solution F was added to every three Wells, gently blown and mixed, and transferred to cell cryopreservation tubes (1 mL per tube).
(2) The labeled information was put into the program cooling box, transferred to the refrigerator at -80 ℃, and stored in a liquid nitrogen tank after 48 hours. Or put it in the 4 ℃ refrigerator for 40 min, put it in the -20 ℃ refrigerator for 2 h, move it to the -80 ℃ refrigerator, and put it in the liquid nitrogen tank after 48 h.

V. Organoid resuscitation and culture
1. Experimental materials:
(1) Materials to be prepared: matrix glue (melt in 4 ℃ refrigerator 24 hours in advance), 15 mL EP tube, water bath, pipettes gun, 24-well cell culture plate, ice box.
(2) Kit reagent: organoid passage culture buffer G (4 ℃), human laryngeal cancer organoid culture medium A.
2. Resuscitation culture of organoids:
(1) The frozen organoids were removed from the low temperature environment and quickly thawed in a 37 ℃ water bath. During the process of water bath melting, it was necessary to gently shake the frozen tube to ensure that the frozen solution was completely thawed in a short time. The thawed organoids were quickly transferred to a 15 mLEP tube, gently blown by a pipettes gun for 6-8 times, and centrifuged at 300 g for 5 min to discard the supernatant. An appropriate amount of passage buffer G was added and resuspended, transferred to a 1.5 mL EP tube and centrifuged at 300 g for 5 min, and the supernatant was discarded.
(2) 100 uL of Matrigel was added to each cryopreservation tube and resuspended. The 24-well cell culture plate was dispensed at 25 ul-30 uL/ well, and the matrigel was maintained at 0-4 ℃ throughout the operation. The cell culture plate was placed in A 37 ℃ incubator for 10-15 min, and after the matrix gel solidified, 500-750 uL of human laryngeal cancer organoid medium A (preheated at 37 ℃) was added to each well for culture.

Storage Temp.

Stored at 4 ° C, valid for 3 months; Stored at -20 ° C, valid for 1 year, see label for details.

AntBio is a biotechnology group company dedicated to serving life sciences, aiming to help scientists accelerate research and improve work efficiency. AntBio provides comprehensive and high-quality reagent tools for basic research, drug development, and diagnosis, including research grade antibodies, proteins, biochemical reagents, and assay kits. These research tools are widely used in different segments of life science research. The group company currently consists of three brands, Absin, Starter-Bio and UA-Bio.